EP1608744A2 - Subtilases - Google Patents

Subtilases

Info

Publication number
EP1608744A2
EP1608744A2 EP04722236A EP04722236A EP1608744A2 EP 1608744 A2 EP1608744 A2 EP 1608744A2 EP 04722236 A EP04722236 A EP 04722236A EP 04722236 A EP04722236 A EP 04722236A EP 1608744 A2 EP1608744 A2 EP 1608744A2
Authority
EP
European Patent Office
Prior art keywords
atom
subtilase
variant
parent
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP04722236A
Other languages
German (de)
French (fr)
Inventor
Allan Svendsen
Stefan Minning
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Novozymes AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novozymes AS filed Critical Novozymes AS
Priority to EP09152523A priority Critical patent/EP2163616A3/en
Publication of EP1608744A2 publication Critical patent/EP1608744A2/en
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the present invention relates to JP170 and BPN' like subtilases and to methods of construction such variants with altered properties, such as stability (e.g. thermostability or storage stability), Ca 2+ dependency, pH dependent activity, improved performance in washing and cleaning applications.
  • stability e.g. thermostability or storage stability
  • Ca 2+ dependency e.g. Ca 2+ dependency
  • pH dependent activity e.g. Ca 2+ dependency
  • improved performance in washing and cleaning applications e.g. thermostability or storage stability
  • Enzymes have been used within the detergent industry as part of washing formula- tions for more than 30 years. Proteases are from a commercial perspective the most relevant enzyme in such formulations, but other enzymes including lipases, amylases, cellu- lases or mixtures of enzymes are also often used.
  • proteases w ith altered properties, such as i ncreased activity at low tempera- tures, increased thermostability, increased specific activity at a given pH, altered Ca 2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc.
  • properties such as i ncreased activity at low tempera- tures, increased thermostability, increased specific activity at a given pH, altered Ca 2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc.
  • proteases with altered properties include both discovery of naturally occurring proteases, i.e. so called wild-type proteases but also alteration of well-known proteases by e.g. genetic manipulation of the nucleic acid sequence encoding said proteases.
  • Knowledge of the relationship between the three-dimensional structure and the function of a protein has improved the ability to evaluate which areas of a protein to alter to affect a specific characteristic of the protein.
  • subtilases One family of p roteases, which are often u sed i n d etergents, a re the subtilases. This family has previously been further grouped into 6 different sub-groups by Siezen RJ and Leunissen JAM, 1997, Protein Science, 6, 501-523.
  • One of these sub-groups is the Subtilisin family which includes subtilases such as BPN', subtilisin 309 (SAVINASE ® , NOVOZYMES A/S), subtilisin Carlsberg (ALCALASE ® , NOVOZYMES A/S), subtilisin S41 (a subtilase from the psychrophilic Antarctic Bacillus TA41 , Davail S et al.
  • s ubtilisin S 39 (a s ubtilase from the psychrophilic Antarctic Bacillus TA39, Narinx E et al. 1997, Protein Engineering, 10 (11 ), pp. 1271-1279) and TY145 (a subtilase from Bacillus sp. TY145, NCIMB 40339 described in WO 92/17577).
  • subtilases belonging to the Subtilisin subgroup were made based on primary sequence alignments, with only little consideration of three-dimensional structure.
  • modelling of the three-dimensional structure of one subtilase on the basis of the three-dimensional structure of another subtilase may result in an incorrect three-dimensional model structure because of structural differences.
  • subtilisin BPN' that was used by Siezen and Leunis- sen
  • the three-dimensional structure of subtilase TY145 have been elucidated and it was found that there are several differences between this and the three-dimensional structure of BPN' also belonging to the Subtilisin subgroup of subtilases (PCT/DK2004/000066).
  • subtilase JP170 and subtilases similar to JP170 are already known in the art, but the three-dimensional structure has not been disclosed for such subtilases.
  • the JP170 subtilase was described as protease A in WO 88/01293 to Novozymes.
  • Figure 1 shows an alignment of three JP170 type proteases: (a) SD-521 (EP 1 209 233), (b) protease Y a (WO 99/67370), and (c) JP170 (WO 98/56927, mature sequence from Appendix 1 ).
  • Figure 2 shows a superposition of the 3D structures of the proteases JP170 and Savinase ( BLSAVI), with i ndication of calcium b inding s ites.
  • I n the figure J P170 i s i ndicated in light grey with three ion-binding sites, and Savinase in a dark structure with two ion-binding sites.
  • Figure 3 shows a matrix of homology between amino acid sequences of subtilases pertaining to various subtilase subgroups.
  • the sequences are identified by sequence database accession numbers and their derivation. 1 : aam50084; Subtilase derived from Bacillus sp. strain SD-521 2: aaw89547; Subtilase derived from Bacillus sp. JP170 3: q45681 ; Subtilase derived from B. subtilis (BSTA41 )
  • sequences 6 to 11 belong to the "true subtilisins” or I-S1 type
  • sequences 12 to 17 belong to the "highly alkaline” subtilisins or I-S2 type. From Fig. 3 it is clear that these types are quite distinct.
  • Figure 4 shows a three-dimensional alignment of the subtilases: (1 ) Ty145; (2) BPN'; (3) Savinase; and (4) JP170.
  • 3D sequences is meant that the position of homologous residues are chosen by superposition of the 3D structures and subsequently the amino acid sequences are aligned based on these homologous positions.
  • APPENDIX 1 s hows the structural coordinates for the solved crystal structure of JP170.
  • Subtilase JP170 (SEQ ID NO:1) Subtilase Y (SEQ ID NO:2) Subtilase SD-521 (SEQ ID NO:3) Subtilase BPN" (SEQ ID NO:4) Partiel sequence (SEQ ID NO:5) Partiel sequence (SEQ ID NO:6) Subtilase TY145 (SEQ ID NO:7)
  • subtilase JP170 This subtilase has large structural differences to the structures of the subtilisins BPN' and TY145.
  • subtilases having a JP170 type structure and subtilases having a BPN' type structure to obtain variants with improved properties.
  • the variants have altered properties, such as in- creased activity at low temperatures, increased thermostability, increased specific activity at a given pH, altered Ca 2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc.
  • the object of the present invention is to provide a method for constructing subtilases having altered properties, in particular to provide a method for constructing subtilases having altered properties as described above.
  • the present invention relates to a method for constructing a variant of a parent subtilase, wherein the variant has at least one altered property as compared to said parent subtilase, which method comprises: a) analyzing the three-dimensional structure of the subtilase to identify, on the basis of an evaluation of structural considerations in relation to a JP170 three dimensional structure, at least one amino acid residue or at least one structural region of the subtilase, which is of relevance for altering said property; b) modifying the DNA of the polynucleotide encoding the parent to construct a polynucleotide encoding a variant subtilase, which in comparison to the parent sub- tilase, has been modified by deletion, substitution or insertion of the amino acid residue or structural part identified in i) so as to alter said property; c) expressing the variant subtilase in a suitable host, and d) testing the resulting subtilase variant for said property.
  • the invention relates to a method of producing a subtilase variant, wherein the variant has at least one altered property as compared to a parent subtilase, which method comprises: a) producing a model structure of the parent subtilase on the three-dimensional structure of BPN', TY145 or JP170; or producing an actually determined three- dimensional structure of the parent subtilase, b) comparing the model or actual three-dimensional structure of the parent subtilase to the JP170 structure by superimposing the structures through matching the CA, CB, C, O, and N atoms of the active site residues, c) identifying on the basis of the comparison in step b) at least one structural part of the parent subtilase, wherein an alteration in said structural part is predicted to re- suit in an altered property; d) modifying the n ucleic a cid sequence e ncoding the p arent subtilase to p roduce a nucleic acid sequence encoding at
  • modification of the parent subti- lase in certain regions and/or positions is expected to confer a particular effect to the thus produced subtilase variant
  • modification of the parent subtilase in any of such regions may also give rise to any other of the above-mentioned effects.
  • any of the regions and/or positions mentioned as being of particular interest with respect to, e.g., improved thermostability may also give rise to, e.g., higher activity at a lower pH, an altered pH optimum, or increased specific activity, such as increased pepti- dase activity.
  • variants of a subtilase t_he DNA encoding such variants and methods of preparing the variants.
  • Still further aspect s of the present invention relates to the use of the variants for various industrial purposes, in particular as an additive in detergent compositions.
  • Other aspects of the present invent tion will be apparent from the below description as well as from the appended claims.
  • subtilases refer to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523.
  • Serine proteases or serine peptidases is a subgroup of proteases characterised by having a serine in the active site, which forms a covalent adduct with the substrate.
  • subtilases (and the serine proteases) are characterised by having two active site amino acid residues apart from the serine, namely a histidine and an aspartic acid residue.
  • Subtilases are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases.
  • the subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
  • the Subtilisin family (EC 3.4.21.62) may be further divided into 3 sub-groups, i.e. I-
  • subtilisins S1 ("true” subtilisins), I-S2 (highly alkaline proteases) and intracellular subtilisins. Definitions or grouping of enzymes may vary or change, however, in the context of the present invention the above division of subtilases into sub-division or sub-groups shall be understood as those described by Siezen et al., Protein Engng. 4 (1991 ) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523.
  • parent is in the context of the present invention to be understood as a protein, which is modified to create a protein variant.
  • the parent protein may be a naturally occurring (wild-type) polypeptide or it may be a variant thereof prepared by any suitable means.
  • the parent protein may be a variant of a naturally occurring protein which has been modified by substitution, chemical modification, deletion or truncation of one or more amino acid residues, or by addition or insertion of one or more amino acid residues to the amino acid sequence, of a naturally-occurring polypeptide.
  • parent subtilase refers to a subtilase which is modified to create a subtilase variant.
  • variant is in the context of the present invention to be understood as a protein which has been modified as compared to a parent protein at one or more amino acid residues.
  • modification(s) or “modified” is in the context of the present invention to be understood as to include chemical modification of a protein as well as genetic manipulation of the DNA encoding a protein.
  • the modification(s) may be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest.
  • modified protein e.g. "modified subtilase”
  • modified protein e.g. subtilase
  • subtilase is to be understood a s a protein which contains modification(s) compared to a parent p rotein, e.g. subtilase.
  • position is in the context of the present invention to be understood as the number of an amino acid in a peptide or polypeptide when counting from the N- terminal end of said peptide/polypeptide.
  • the position numbers used in the present invention refer to different subtilases depending on which subgroup the subtilase belongs to. As m entioned above the alkaline subtilases KP43, KP1790, KP9860, Y, SD-521 and E1 belong to the JP170 subgroup, based on sequence homology. Due to the exten- sive homology only subtilase Ya and SD-521 are in Fig. 1 aligned with JP170.
  • the JP170 subtilase, Y subtilase and SD-521 subtilase are numbered according to SEQ ID NO:1 , SEQ ID NO:2 and SEQ ID NO:3, respectively.
  • the invention is not limited to variants of these particular subtilases but extends to parent subtilases, especially of the JP170 type, containing amino acid residues at positions which are "equivalent" to the particular identified residues in the JP170 subtilase.
  • a residue (amino acid) position of a JP170 type subtilase is equivalent to a residue (position) of the JP170 subtilase, if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in the JP170 subtilase (i.e., having the same or similar functional capacity to combine, react, or interact chemically).
  • the amino acid sequence of a precursor protease is directly compared to the JP170 subtilase primary sequence by aligning t he a mino a cid s equence of an i solated o r p arent wild type e nzyme with a suitable well-known (standard) enzyme of the same group or class of enzymes to define a frame of reference.
  • a suitable well-known (standard) enzyme of the same group or class of enzymes to define a frame of reference.
  • This type of numbering has been used in numerous patent applications relating to subtilisins of the I-S1 and I-S2 subgroups with subtilisin BPN' as the standard subtilisin. If nothing else is indicated herein, in the present instance the JP170 subtilase has been chosen as standard.
  • the 3D structure based a lignment i n F ig. 1 h as been provided.
  • the amino acid sequence of a precursor JP170 type subtilase may be directly correlated to the JP170 primary sequence.
  • the (3D based) position corresponding to a position in JP170 is found by i) identifying the JP170 type subtilase from the alignment of Fig.
  • the alignment may define a number of deletions and insertions in relation to the sequence of JP170. In the alignment deletions are indicated by asterixes ( * ) in the referenced sequence, and the referenced enzyme will be considered to have a gap at thie position in question.
  • Insertions are indicated by asterixes (*) in the JP170 sequence, and the positions in the referenced enzyme are given as the position number of the last amino acid residue where a corresponding amino acid residue exists in the standard enzyme with a lower case letter appended in alphabetical order, e.g. 82a, 82b, 82c, 82d.
  • the referenced enzyme contains a N- or C-terminal extension in comparison to JP170; an N-terminal extension is given the position number 0a, 0b, etc. in the di rection of the N-terminal; and a C-terminal extension will be given either the position number of the C-terminal amino acid residue of JP170 with a lower case letter appended in alphabetical order, or simply a continued consecutive numbering.
  • JP170 type subtilases are numbered by reference to the positions of the JP170 subtilase (SEQ ID NO: 1 ) as provided in Fig. 1. The position is then indicated as "corresponding to JP170".
  • Subtilases belonging to the BPN' subgroup refers to the positions of Subtilisin Novo
  • Subtilases belonging to the TY145 subgroup refers to the positions of the TY145 subtilase (SEQ ID NO:7), see also PCT/DK2004/000066.
  • subtilases Despite the great homology of the subtilases described above the inventors of the present invention have elucidated the three-dimensional structure of JP170, SEQ ID NO:1 by X-ray crystallography and found that there are several differences between this and the three-dimensional structure of BPN'. The inventors of the present invention have further compared the sequence homology of subtilases belonging to the Subtilisin subgroup. This is shown in Figure 3 of the present invention.
  • JP170 type subtilases become a separate subgroup in addition to the subgroups of BPN' subtilases and TY145 subtilases PCT/DK2004/000066. JP170 type subtilases
  • JP170 subtilase or "JP170 type subtilase” should in the context of the present invention be understood as a subtilase belonging to the Subtilisin group according to Siezen et al. Protein Science 6 (1997) 501-523 and which has at least 58% homology to JP170, SEQ ID NO:1.
  • JP170 type subtilase may have at least 60% homology to SEQ ID NO:1 , such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to JP17O, i.e. to SEQ ID NO:1.
  • the alkaline proteases KP43, KP1790, KP9860, Prote- ase Ya, Protease E-1 and SD-521 are subtilases belonging to the JP170 subgroup of subtilases.
  • a JP170 subtilase suitable for the purpose described herein m ay be a subtilase homologous to the three-dimensional structure of JP170, i.e. it may be homologous to the three-dimensional structure defined by the structure coordinates in Appendix 1.
  • a set of structure coordinates for a protein or a portion thereof is a relative set of points that define a shape in three dimensions, it is possible that an entirely different set of coordinates could define an identical or a similar shape.
  • slight variations in the individual coordinates may have little or no effect on the overall shape. These variations in coordinates may be generated because of mathematical manipulations of the structure coordinates.
  • the structure coordinates of JP170 may be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
  • said variations may be due to differences in the primary amino acid sequence.
  • the standard error may typically be measured as the root mean square deviation of e.g. conserved backbone residues, where the term "root mean square deviation” (RMS) means the square root of the arithmetic mean of the squares of the deviations from the mean.
  • RMS root mean square deviation
  • similarities in 3D structure of different subtilases can be described by the content of homologous structural elements, and/or the similarity in amino acid or DNA sequence. For sequences having no deletions or insertions a RMS for the CA carbon atoms can be calculated.
  • a JP170 type subtilase is further characterised as comprising the following structural characteristics: a) a twisted beta-sheet with 7 strands, b) six alpha helices, c) at least three ion-binding sites, and not comprising the Strong and Weak ion-binding site of the BPN' like subtilases
  • the isolated nucleic acid sequence encoding a JP170 subtilase of the invention hybridizes with a complementary strand of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:1 preferably under low stringency conditions, at least under medium stringency conditions, at least under medium/high stringency conditions, at least under high stringency conditions, at least under very high stringency conditions.
  • Suitable experimental conditions for determining hybridization at low, medium, or high stringency conditions between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the filter in a solution of 5 x SSC, 5 x Denhardt's solution (Sambrook et al. 1989), 0.5 % SDS and 100 ⁇ g/ml of denatured sonicated salmon sperm DNA (Sambrook et al.
  • the filter is then washed twice for 30 minutes in 2 x SSC, 0.5 % SDS at least * 55°C (low stringency), more preferably at least 60°C (medium stringency), still more pref- erably at least 65°C (medium/high stringency), even more preferably at least 70° C (high stringency), and even more preferably at least 75°C (very high stringency).
  • a BPN' subtilase or BPN' type subtilase is in the context of the present invention to be understood as a subtilase belonging to the Subtilisin group according Siezen et al. Siezen et al. Protein Science 6 (1997) 501-523 and which has at least 61% homology to SEQ ID NO:4.
  • a BPN' subtilase may have at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to BPN', i.e. to SEQ ID NO:4.
  • isolated nucleic acid sequence encoding a BPN' subtilase of the invention hybridizes with a complementary strand of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:4 preferably under low stringency conditions, at least under medium stringency conditions, at least under medium/high stringency conditions, at least under high stringency conditions, at least under very high stringency conditions.
  • a BPN' subtilase suitable for the purpose described herein may be a subtilase homologous to the three-dimensional structure of BPN' as defined by the structure coordinates given in PDB Nos. 1SBT and 1GNS (Protein Data Bank), or one of the several other structures of BPN' that are accessible from the Protein Data Bank. Variations between homologous structures may occur for several reasons as described above.
  • a BPN' subtilase within the context of the present invention is to be understood as any subtilase having the structural characteristics pertaining to the BPN' subtilases as described above, and in addition such subtilases do preferably not have further structural characteristics which are not present in the BPN' subtilases as de- scribed herein.
  • a BPN' type subtilase has two ion- binding sites.
  • a BPN' like subtilase may, in the context of the present invention, belong to branch l-S of the subtilisins i.e. to branch I-S1 , the "true” subtilisins or I-S2, the highly alkaline proteases (Siezen et al., Protein Engng. 4 (1991 ) 719-737).
  • subtilisin 309 subtilisin 309
  • SAVI- NASE ® subtilisin ®
  • subtilisin Carisberg ALCALASE ®
  • NOVOZYMES A/S subtilisin Carisberg
  • the Weak calcium-binding site was shown to be a Na (sodium) binding site when the calcium concentration in the crystallization medium was low.
  • ion- binding sites instead of calcium-binding sites.
  • a TY145 subtilase or TY145 type subtilase is in the context of the present invention to be understood as a subtilase which has at least 63% homology to SEQ ID NO:7.
  • said TY145 subtilase may have at least 65%, such as at least 70%, at least 74%, at least 80%, at least 83%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to TY145, i.e. to SEQ ID NO:7.
  • a TY145 subtilase suitable for the purpose described herein may be a subtilase homologous to the three-dimensional structure of TY145 as defined by the structure coordinates given in PCT/DK2004/000066. Variations between homologous structures may occur for several reasons as described above.
  • a TY145 subtilase within the context of the present invention is to be understood as any subtilase having the structural characteristics pertaining to the TY145 subtilases as described above, and in addition such subtilases do preferably not have further structural characteristics which are not present in the TY145 subtilases as described herein.
  • a TY145 subtilase further comprises the following structural characteristics: a) a twisted beta-sheet with 7 strands, b) six alpha helices, c) at least three ion-binding sites, wherein the Strong ion-binding site of the BPN' type sub- tilases is not present,
  • Examples of s ubtilases of the TY145 type i include the TY145 s ubtilase, the psychrophilic subtilisin protease S41 derived from the Antarctic Bacillus TA41 , herein also called TA41 subtilase (Davail S et al., 1994, J. Biol. Chem., 269, 17448-17453), and the psychrophilic subtilisin protease S39 derived from the Antarctic Bacillus TA39, herein also called TA39 subtilase (Narinx E et al., 1997, Protein Engineering, 10 (11 ), 1271-1279).
  • TA41 subtilase the psychrophilic subtilisin protease S41 derived from the Antarctic Bacillus TA41
  • TA41 subtilase the psychrophilic subtilisin protease S39 derived from the Antarctic Bacillus TA39
  • the JP170 subtilase was used to elucidate the three-dimensional structure forming the basis for the present invention.
  • JP170 was solved in accordance with the principle for x-ray crystallographic methods, for example, as given in X-Ray Structure Determination , Stout, G.K. and Jensen, L.H., John Wiley & Sons, Inc. NY, 1989.
  • the protease coordinates has a chain identification called A, whereas the CI2 inhibitor is called B, the calcium ions are called C, and the water is W.
  • the positions of the mentioned residues refer to the sequence of JP170 as disclosed in SEQ ID NO:1.
  • the JP170 structure consists of two domains, a catalytic domain and a C-terminal domain.
  • the structure of the catalytic domain shows the same overall fold as found in the S8 family of subtilisins.
  • the structure comprises a twisted beta-sheet with 7 strands arranged in the following sequential order S2, S3, S1 , S4, S5, S6, S7.
  • H1 contains residues 9-17
  • H 2 contains residues 68-76
  • H3 contains residues 1 10-1 19
  • H 4 contains residues 139-150
  • H5 contains residues 253-273
  • H6 contains residues 281- 291.
  • the C-terminal domain comprises a strand motif, a so called "beta sandwich” consisting of sheets a and b.
  • the sheet in this domain is combined of strands in an anti- parallel fashion, whereas the strand in the catalytic domain is combined in parallel.
  • the sequential order of the strands can be denoted as: S1a-S1b-S3a-S3b-S4b-S4a-S2b-S2a with the beta sandwich organised as to the two sheets S1a, S3a, S4a, S2a and S1 b, S3b, S4b, S2b.
  • the JP170 subtilases were found to lack the well-known Strong and Weak ion- binding sites of the BPN' subtilases. However, the JP170 subtilases have three ion-binding sites which are not present in the BPN' subtilisin structures. This can be seen in the struc- tural alignment presented in Fig. 2. These three ion-binding sites are hereinafter referred to as Site 1 , which is placed in the catalytic domain, and Site 2 and 3 which are placed in the non-catalytic C-terminal domain.
  • Site 2 - calcium atom named A603 CA
  • Site 3 - calcium atom named A602 CA in the PDB table (Appendix 1).
  • the position of an ion-binding site can be defined by the distance to four specific atoms in the core structure.
  • the distance from the ion-binding site to the c-alpha atoms of the three active site residues has been chosen.
  • the residues Ser, His and Asp in the active site are highly conserved. In JP170 they are Asp30, His68 and Ser254.
  • the fourth distance chosen is the distance to the c-alpha atom of the arnino acid residue coming first after the active site serine residue in the sequence (herein after called "next to Ser"); in the 3D structure of JP170 it is Met255. o
  • the distance between: a) ion-binding site 1 and i) Asp c-alpha atom is 26.70-28.7 ⁇ A, ii) His c-alpha atom is 22.10-24.10A, iii) Ser c-alpha atom is 16.95-18.95A, iv) next to Ser c-alpha atom is 15.30-17.30A, b) ion-binding site 2 and i) Asp c-alpha atom is 33.50-35.5 ⁇ A, ii) His c-alpha atom is 37-39A, iii) Ser c-alpha atom is 29.40-31.40A, iv) next to Ser c-alpha atom is 30.70-32.70A, c) ion-binding site 3 and i) Asp c-alpha atom is 41.50-43.50A, ii) His c-alpha
  • these distances may vary from one subtilase to the other.
  • the present distances are given with a calcium ion in the structure. If a sodium ion was bound instead the distances would be shifted a little bit.
  • the distances can vary ⁇ 0.8 ⁇ A, pref- erably ⁇ 0.7 ⁇ A, ⁇ 0.6 ⁇ A, ⁇ 0.5 ⁇ A, ⁇ 0.4 ⁇ A, or most preferably ⁇ 0.3 ⁇ A.
  • the peptide structure circumscribing ion- binding site 1 up to a distance of 10 A from the metal ion is composed of the amino acid residues placed in positions 183-189, 191-204 and 224-225.
  • the peptide structure circumscribing ion-binding site 2 up to a distance of 10 A from the metal ion is composed of residues 378-393.
  • the peptide structure circumscribing ion-binding site 3 up to a distance of 10 A from the metal ion is composed of residues 348, 350, 352, 363-370, 380-383, 391-400 and 414- 420.
  • the structure of the JP170 like subtilases can be divided into a "core subtilase-like” region, an "intermediate” region and a "nonhomologous” region.
  • the active site can be found in the core subtilase-like region, which is structurally closely related to the BPN' structures.
  • the core subtilase-like region is composed of residues 17-34, 197-209 and 216-232, and contains the alpha-helix H3 and the central alpha- helix H5 in which the active site serine residue is situated in the N-terminal part.
  • the core subtilase-like region has an RMS lower than 1.2.
  • the intermediate region consists of residues 42-46, 150-186, 245-272 and 278- 296.
  • the intermediate region has an RMS bigger than 1.2 and less than 1.8.
  • the relationships between the three-dimensional structure and functionality are potentially difficult to predict in this region of the JP170 like subtilases.
  • the nonhomologous region consists of residues 1-16, 35-41 , 47-149, 187-196, 210-215, 233-244, 273-277 and 297-316.
  • the nonhomologous region has a RMS h igher than 1.8.
  • the relationships between the three-dimensional structure and functionality are very difficult to predict in this region of the JP170 like subtilases.
  • the loops N79-N82 (I75-V81 ) and G208-D218 (G193-D197) are in contact with a ion-binding site in Savinase, but not in JP170.
  • the loop E185-N199 (D181-N 184) is in contact with a ion-binding site in JP170, but not in Savinase.
  • a good example of the difference is the loop S232-K246 in JP170 which has 15 residues compared to the corresponding BPN' type loop G211-T213 (in Savinase), which has only three residues.
  • the loop folds back to the substrate binding site, especially the P' parts of the substrate binding site.
  • the loop is situated close to the substrate as illustrated by the CI2 inhibitor bound in the 3D structure attached (Ap- pendix l ).
  • loop S232-K246 in JP170 can be described in relation to the four specific residues as described above.
  • the distance from the CA atom of residue W240 in the loop to the CA atoms of the active site residues are: Residue H68 D30 S254 M255
  • distances like these can vary ⁇ 0.8 ⁇ A, preferably ⁇ 0.7 ⁇ A, ⁇ 0.6 ⁇ A, ⁇ 0.5 ⁇ A, ⁇ 0.4 ⁇ A, or most preferably ⁇ 0.3 ⁇ A.
  • distances from the residues of JP170 loop S232-K246 to atoms of the CI2 inhibitor can be calculated. These distances are: from CA atom of W240 to CA atom of R62 in CI2 is 7.49A, from CA atom of F239 to CA atom of R62 in CI2 is 8.39A, from CA atom of S238 to CA atom of R62 in CI2 is 8.42A, from CA atom of S237 to CA atom of R62 in CI2 is 9.44A, from CA atom of S238 to CA atom of E60 in CI2 is 9.42A.
  • the distances from JP170 active site residue S254 to atoms of the CI2 inhibitor, as placed in the 3D coordinates of Appendix 1 are: from CA atom of S254 to CA atom of E60 in CI2 is 5.25A, from CA atom of S254 to CA atom of R62 in CI2 is 11.55A, from CA atom of S254 to CA atom of T58 in CI2 is 7.06A, from CA atom of S254 to CA atom of M59 in CI2 is 4.71 A.
  • the distances can vary ⁇ 0.8 ⁇ A, preferably ⁇ 0.7 ⁇ A, ⁇ 0.6 ⁇ A, ⁇ 0.5 ⁇ A, ⁇ 0.4 ⁇ A, or most preferably ⁇ 0.3 ⁇ A.
  • a preferred JP170 like subtilase variant has a deletion in the region S232-K246, and the subsequent insertion of one or more residues to partly or completely remove the loop.
  • Preferred variants comprises the deletion of L233-S245 + insertion of Asn, deletion of L233-D244 + insertion of Gly or deletion of S232-D244 + insertion of Gly. Similar considerations can be made in respect of differences to the TY145 structure.
  • a model structure of a JP170 type subtilase, a BPN' type subtilase or a TY145 type subtilase can be built using the Homology program or a comparable program, e.g., Modeller (both from Molecular Simulations, Inc., San Diego, CA).
  • the principle is to align the amino acid sequence of a protein for which the 3D structure is known with the amino acid sequence of a protein for which a model 3D structure has to be constructed.
  • the structur- ally conserved regions can then be built on the basis of consensus sequences.
  • loop structures can be inserted, or sequences can be deleted with subsequent bonding of the n ecessary residues using, e .g., the program Homology. S ubsequent relaxing and optimization of the structure should be done using either Homology or another molecular simulation program, e.g., CHARMm from Molecular Simulations.
  • the present invention relates to a method for constructing a variant of a parent subtilase, wherein the variant has at least one altered property as compared to said parent subtilase, which method comprises: a) analyzing the three-dimensional structure of the parent subtilase to identify, on the basis of an evaluation of structural considerations in relation to a JP170 three di- mensional structure, at least one amino acid residue or at least one structural region of the subtilase, which is of relevance for altering said property; b) modifying the DNA of the polynucleotide encoding the parent to construct a polynucleotide encoding a variant subtilase, which in comparison to the parent subtilase, has been modified by deletion, substitution or insertion of the amino acid residue or structural part identified in i) so as to alter said property; c) expressing the variant subtilase
  • the invention relates to a method of producing a subtilase variant, wherein the variant has at least one altered property as compared to a parent subtilase, which method comprises: a) producing a model structure of the parent subtilase on the three-dimensional structure of BPN', TY145 or JP170; or producing an actually determined three- dimensional structure of the parent subtilase, b) comparing the model or actual three-dimensional structure of the parent subtilase to the JP170 structure by superimposing the structures through matching the CA, CB, C, O, and N atoms of the active site residues, c) identifying on the basis of the comparison in step b) at least one structural part of the parent subtilase, wherein an alteration in said structural part is predicted to re- suit in an altered property; d) modifying the n ucleic a cid sequence e ncoding the parent subtilase to produce a nucleic acid sequence encoding at least one deletion or substitution of one or more
  • the present invention thus generally relates to the use of the JP170 structure as provided herein for the identification of desired modifications in subtilases of any of the three subtilisin types, the BPN' types (I-S1 and I-S2 subgroups), the TY145 types and the JP170 types through modelling the 3-D structure of a parent subtilase to the type it belongs to and subsequent comparison thereof to the JP170 3-D structure, or in instances where the 3-D structure of the parent subtilase is actually known by comparison thereof to the JP170 3-D structure.
  • At least one residue in the parent subtilase is selected for modification by substitution, deletion or insertion in order to provide a subtilase variant with altered properties in comparison to the parent subtilase.
  • the parent subtilase may therefore belong to the sub-group I- S1 , preferably selected from the group consisting of ABSS168, BASBPN, BSSDY, and BLSCAR, or functional variants thereof having retained the characteristic of sub-group I- S1.
  • the parent subtilase belongs to the sub-group I-S2, preferably selected from the g roup consisting of BLS147, BLS309, BAPB92, and BYSYAB, or functional variants thereof having retained the characteristic of sub-group I-S2.
  • subtilase belongs to the TY145 type subgroup, preferably selected from the group comprising TY145, protease S41 also called TA41 protease S39 also called TA39 subtilase, etc.
  • subtilase belongs to the JP170 type subgroup, preferably selected from the group comprising JP170, KP43, KP9860, Protease E-1 , Protease Ya, Protease SD-521 , etc.
  • a further embodiment of the invention relates to a method of producing a JP170 type subtilase variant, wherein the variant has at least one altered property as compared to a parent subtilase, which method comprises: a) producing a model structure of the parent JP170 type subtilase on the three- dimensional structure of JP170; or producing an actually determined three- dimensional structure of the parent subtilase, b) comparing the model or actual three-dimensional structure of the parent JP170 type subtilase to the BPN' or TY145 structure by superimposing the structures through matching the CA, CB, C, O, and N atoms of the active site residues, c) identifying on the basis of the comparison in step b) at least one structural part of the parent JP170 type subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent JP170 type subtilase to produce a nucleic acid sequence
  • the invention also comprises the protease variants produced by the above meth- ods.
  • JP170 subtilases As described above the three-dimensional structure of JP170 subtilases as provided in Appendix 1 indicates the presence of three ion-binding sites not present in the BPN' subtilisin structures, thus lacking the Strong and Weak ion-binding site of the BPN' subtilases. Stability of ion-binding sites is important for the functionality of the enzyme. Therefore alterations of the ion-binding sites are likely to result in alterations of the stability of the enzyme.
  • step (c) above identifies amino acid residue positions located at a distance of 1 ⁇ A or less to the ion-binding site of the JP170 type parent, preferably positions located at a distance of 6 A or less.
  • a preferred variant of the present invention has a modification in one or more of the positions located at a distance of 1 ⁇ A to the ion-binding sites of JP170 (SEQ ID NO:1). These positions are:
  • Site 1 183-189 (i.e. positions 183, 184, 185, 186, 187, 188, 189),
  • Site 2 378-393 (i.e. positions 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387,
  • Site 3 348, 350, 352, 363-370 (i.e. positions 363, 364, 365, 366, 367, 368, 369, 370),
  • Preferred variants stabilised in ion-binding site 2 are N390D and N391 D
  • preferred variants stabilised in ion-binding site 3 are G394N,Q,F,Y,S and W392S,N,Q.
  • a variant with improved stability may be obtained by substitution with proline, introduction of a disulfide bond, altering a hydrogen bond contact, altering charge distribution, introduction of a salt bridge, filling in an internal structural cavity with o ne o r m ore a mino acids with b ulkier s ide groups (in e.g. regions which are structurally mobile), substitution of histidine residues with other amino acids, removal of a deamidation site, or by helix capping.
  • the following regions of JP170 have an increased mobility in the crystal structure of the enzyme, and it is presently believed that these regions can be responsible for stability or activity of JP170. Especially thermostabilisation may possibly be obtained by altering the highly mobile regions. Improvements of the enzyme can be obtained by mutation in the below regions and positions. Introducing e.g. larger residues or residues having more atoms in the side chain could increase the stability, or, e.g., introduction of residues having fewer atoms in the side chain could be important for the mobility and thus the activity profile of the enzyme.
  • Two methods extract the highly mobile regions from a 3D structure.
  • One is a molecular dynamics calculation of the isotropic fluctuations by using the program CHARMm from MSI (Molecular Simulations Inc.), and the other is an analysis of the B-factors.
  • the B- factors are listed in Appendix 1 and give a value to the uncertainty of determination of the location of the various atoms of the structure.
  • the uncertainty relates to the mobility of the atoms in the molecules in the crystal lattice. This mobility reflects the thermal motion of the atoms and thus indicates possible sites for thermostabilisation of the enzyme.
  • 131-134 i.e. positions 131 , 132, 133, 134
  • 107-111 i.e. positions 107, 108, 109, 110, 111
  • 337-342 i.e. positions 337, 338, 339, 340, 341 , 342
  • a preferred JP170 subtilase variant of the present invention has been modified in one or more of the above mentioned positions of SEQ ID NO:1.
  • Further preferred variants comprises one or more alterations in the regions 57-60, 66-67, 107-1 1 1 , 236-240, 326-332, 355-360, 372-377, 384-388, 404-41 1.
  • variant W240H.Y and variants modified in the region 355-360 such as variants comprising one or more of the modifications: G355A.S; S356T.N; T357N,Q,D,E,P; T358S; A359S,T,N,Q and S360T.N.
  • Variants modified in the region 355-360 may be produced in accordance with the method for random mutagenesis by use of the DOPE program as described herein. To obtain variants comprising 1-3 modifications in region 355-360 one may introduce the substitutions with the following frequencies: wild-type modified
  • a JP170 variant of the present invention with improved stability, e.g. thermostability, as compared to the parent JP170 subtilase may be obtained by introducing new inter- domain or intra-domain bonds, such as by establishing inter- or intra-domain disulfide bridges.
  • a further aspect of the present invention relates to a method for producing a variant of a parent JP170, wherein step (c) identifies amino acid residue positions in the parent JP170 type subtilase, the modification of which may create at least one disulfide bridge by insertion of or substitution with at least one Cys residue.
  • substitutions are: G21C+A86C, V26C+A265C, G57C+G105C, G74C+A229C, Q111C+N143C, G160C+S170C, A286C+V349C, A27C+A122C, A45C+G78C, V72C+P258C, G78C+A229C, D98C+G104C, Q111C+Y147C, G135C+G167C, R142C+P354C, V144C+A178C, G182C+P217C, A183C+G223C, A195C+Y225C, F271C+P279C, A287C+A430C, A293C
  • a variant with improved stability as compared to the parent subtilase may be obtained by changing the surface charge distribution of the subtilase. For example, when the pH is lowered to about 5 or below histidine residues typically become positively charged and, consequently, unfavorable electrostatic interactions on the protein surface may occur. By engineering the surface charge of the subtilase one may avoid such unfavorable electrostatic interactions that in turn lead to a higher stability of the subtilase.
  • Charged amino acid residues are (a) positively charged: Lys, Arg, His (pH ⁇ 5), Tyr (pH>9) and Cys (pH>10) and (b) negatively charged: Asp and Glu.
  • a further aspect of the present invention relates to method for constructing a variant of a parent subtilase, the method comprising: a) identifying, on the surface of the parent subtilase, preferably a JP170 like or a BPN' like subtilase, at least one amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His; b) substituting, on the surface of the parent subtilase, at least one amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His with an uncharged amino acid residue; c) optionally repeating steps a) and b) recursively; d) optionally, making alterations each of which is an insertion, a deletion or a substitution of an amino acid residue at one or more positions other than b); e) preparing the variant resulting from steps a) - d); f) testing the stability of said variant; and g) optionally repeating steps a) - f
  • the above method may be employed. Again, compared to the above method, the only difference being steps a) and b) which, in this case, read: a) identifying, on the surface of the parent subtilase, at least one charged amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His; b) substituting, on the surface of the parent subtilase, at least one charged amino acid residue identified in step a) with an amino acid residue having an opposite charge.
  • Lys or His may be substituted with Asp or Glu; Lys may be substituted with Asp or Glu; and His may be substituted with Asp or Glu.
  • the surface accessible area are measured using the DSSP pro- gram (Kabsch and Sander, Biopolymers (1983), 22, 2577-2637). All residues having a surface accessibilty higher than 0 0, 0.10, 0.20, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55 or 0.60 are regarded a surface residue.
  • N79, N316, L381 , K246, K9, K313 and K83 are amino acid residues found on the surface of JP170 using the above method.
  • substitutions N79D, N316D and L381 D are of particular interest for stabilisation by introduction of salt bridges
  • substitutions K246R, K9R, K313R and K83R are of particular interest for the stabilisation at high pH.
  • thermostability of a subtilase can be obtained by subjecting the subtilase in question to analysis for secondary structure, identifying residues in the subtilase having dihedral angles ⁇ (phi) and ⁇ (psi) confined to the intervals [-90° ⁇ -40° and -180° ⁇ 180°], preferably the intervals [-90° ⁇ -40° and 120° ⁇ 180°] or [-90° ⁇ -40° and -50° ⁇ 10°] and excluding residues located in regions in which the subtilase is characterized by possessing ⁇ - helical or ⁇ -sheet structure.
  • proline residues are generally not compatible with ⁇ -helical and ⁇ -sheet secondary con- formations.
  • a proline residue is not already at the identified position(s)
  • the naturally occurring amino acid residue is substituted with a proline residue, preferably by site directed mutagenesis applied on a gene encoding the subtilase in question.
  • proline residues can advantageously be intro- quizzed at positions 22, 44, 110, 139, 140, 166, 198, 201 , 203, 231 , 282, 356, 357 and 378.
  • a preferred JP170 variant has one or more of the substitutions: Q22P, E44P, L110P, T139P, D140P, S166P I198P, V201P, Q203P, S231P, S282P, S356P, T357P and K378P.
  • variants comprising one or more of: E44P, Q203P and S356P.
  • JP170 subtilases differ greatly from the BPN' like subtilases in having a long apparently non-catalytic C-terminal.
  • a possible truncation of JP170 is the removal of approx. 115 residues including two ion-binding sites, which can be obtained by deletion of or within the region 311-433, which is the non-catalytic C-terminal.
  • Preferred deletions com- prises the regions 317-433 or 315-433.
  • the new C-terminal will be within the region of 311-325.
  • the deletion can be optimised with additional substitutions, such as one or more of L283N.Q; A290S.N and W306H,Y,K.
  • Preferred truncations comprise: a) deletion of region 317-433 and the substitutions L283N + A290S + W306H, b) deletion of region 315-433 and the substitutions L283N + A290S + W306H.
  • the substrate binding site is identified by the residues in contact with a substrate model, such as the CI2 inhibitor.
  • a substrate model such as the CI2 inhibitor.
  • the 3D structure coordinates of the JP170 subtilase with CI2 bound in the active site are provided in Appendix 1. Without being limited to any theory, it is presently believed that binding between a substrate and an enzyme is supported by favorable interactions found within a sphere 10 A from the substrate molecule. Examples of such favorable bonds are hydrogen bonds, strong electrostatic interaction and/or hydrophobic interactions.
  • residues of the JP170 subtilase are within a distance of 1 ⁇ A from the CI2 inhibitor which is bound to the substrate binding site. These residues are thus believed to be involved in interactions with said substrate: 29-32, (i.e. residues 29, 30, 31 , 32) 64-72, (i.e. residues 64, 65, 66, 67, 68, 69, 70, 71 , 72)
  • 100-110 (i.e. residues 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110) 113-114, 127-136, (i.e. residues 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136) 138-141 , (i.e. residues 138, 139, 140, 141 ) 144, 157, 174,
  • a variant comprises a modification in one or more of the above mentioned positions.
  • a preferred variant is W129L. JP170 with extra ion-binding site
  • the Strong ion-binding site from the BPN' subtilases can be transplanted into JP170 (or other subtilases in JP170 subgroup) by deletion of N79-N82 and subsequent insertion of LNNSIGV (SEQ ID NO:5), followed by the substitution A45D.N and optionally the substitutions E44P,T and/or R47Q.
  • JP170 or other JP170 type subtilases can be removed with guidance from information about the three-dimensional structures of other related subtilases, such as a BPN' type subtilase, such as Savinase or BPN', and a
  • Removal of ion-binding site 1 can be done by deletion of N186-N199 and subse- quent insertion of at least three amino acid residues - or stated differently by the substitution of a region comprising from 3 to 6 amino acid residues for the region comprising 14 amino acid residues in positions 186 to 199, preferably the sequence of the substituting region is SSN (SEQ ID NO:6).
  • SSN SEQ ID NO:6
  • substitutions I7Q and V3Y is further added.
  • the ion-binding site 1 can be removed from a wild-type JP170 subtilase or a JP170 subtilase truncated as described above.
  • JP170 type subti- lases and TY145 type subtilases can be used to remove the Strong and Weak ion-binding sites in BPN' type subtilases, or the ion-binding sites in TY145 type subtilases may be removed on the b asis of structural i nformation a bout the J P170 and BPN' types of s ubtilases.
  • the removal can be done by altering the loops A194-L196 (weak ion-binding site) and L75-L82 (strong ion-binding site) either by a) insertion or deletion of a number of amino acid residues in the loops or b) by deletion of the entire loop or part of the loop and subsequent insertion of a number of residues from a corresponding loop of a JP17J0 or TY145 like subtilase.
  • the ion-binding sites of Savinase can be removed by either i) full or partial deletion of the region A194-L196 (BPN' numbers) and insertion of three or more residues chosen from JP170 positions P209-P217, and ii) full or partial deletion of the region L75-L82 (BPN' numbers) and insertion of at least one residue chosen from TY145 positions H83-Y92 or i) full or partial deletion of the region A194-L196 (BPN 1 numbers) and insertion of three or more residues chosen from JP170 positions P209-P217 and ii) full or partial deletion of the region L75-L82 (BPN' numbers) and insertion of at least one residues chosen from JP170 positions N79-K83.
  • JP170 type subtilase protease it may be advantageous to substitute or delete critical oxidation sites, such as methionines, with other amino acid residues which are not subject to oxidation.
  • the present invention relates to an RP-II pro- tease variant, in which one or more amino acid residues susceptible to oxidation, especially m ethionine residues exposed to the surface of the molecule, is/are d eleted or replaced with another amino acid residue less susceptible to oxidation.
  • the amino acid residue less susceptible to oxidation may for instance be selected from the group consisting of A, E, N, Q, I, L, S and K. Specific such variants comprises at least one of the deletions or substitutions
  • the invention consequently further relates to a subtilase, in which the modificatioins indicated above are either or both residues of any of the Asn-Gly sequence appearing in the amino acid sequence of the parent RP-II protease is/are deleted or substituted with a residue of a different amino acid.
  • the Asn and/or Gly residue may, for instance, be substituted with a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y.
  • any of the Asn or Gly residues of the Asn-Gly occupying positions 66-67, 134-135 and/or 375-376 of the SD-521 and Ya protease; and positions 66-67, 134- 135, 301-302 and/or 375-376 of the JP170 protease, may be deleted or substituted with a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y. (positions are indicated in relation to the JP170 protease as indicated in Fig. 1 ).
  • JP170 Specific variants of JP170 are: N66f,A,Q,S,P,T,Y ⁇ ; G67f ,A,Q,S,P,T,Y ⁇ ; N134 ⁇ * ,A,Q,S,P,T,Y ⁇ ; G135 ⁇ * ,A,Q,S,P,T,Y ⁇ ; N301 ⁇ * ,A,Q,S,P,T,Y ⁇ ; G302f ,A,Q,S,P,T,Y ⁇ ;
  • SD-521 and Ya proteases are: N66 ⁇ *,A,Q,S,P,T,Y ⁇ ; G67 ⁇ * ,A,Q,S,P,T,Y ⁇ ; N134 ⁇ * ,A,Q,S,P,T,Y ⁇ ; G135f ,A,Q,S,P,T,Y ⁇ ; and N375 ⁇ *,A,Q,S,P,T,Y ⁇ ; G376 ⁇ * ,A,Q,S,P,T,Y ⁇ , and combinations thereof as indicated above.
  • JP170 tyrosine residues may be modified in positions: 20, 54, 1 18, 137, 147, 194, 225, 247, 249, 334, 379, 388, 411 , and 418.
  • the tyrosine residues may be modified in positions: 17, 20, 54, 137, 147, 187, 243, 247, 249, 299, 319, 334, 361 , 379, 386, 388, 411 , and 418.
  • the invention thus relates to the variants: Y17 ⁇ F,W ⁇ , Y20 ⁇ F,W ⁇ ,
  • JP170 type subtilases JP170 type subtilases.
  • the invention relates to JP170 type subtilase variants comprising one or more of the following substitutions: For the SD-521 a nd Ya proteases positions 1 18, 1 29, 240, 306, 350, and 392; and for the JP170 protease positions 129, 240, 306, 350, and 392.
  • the invention relates to a JP170 variant comprising one or more of the following substitutions W129 ⁇ F,T,Q,G ⁇ , W240 ⁇ F,T,Q,G ⁇ , W306 ⁇ F,T,Q,G ⁇ , W350 ⁇ F,T,Q,G ⁇ , and W392 ⁇ F,T,Q,G ⁇ .
  • the present invention also encompasses any of the above mentioned subtilase variants in combination with any other modification to the amino acid sequence thereof. Especially combinations with other modifications known in the art to provide improved properties to the enzyme are envisaged.
  • the subtilase variants i.e. the JP170 and BPN' variants of the present invention may be produced by any known method within the art and the present invention also relates to nucleic acid encoding a subtilase variant of the present invention, a DNA construct comprising said nucleic acid and a host cell comprising said nucleic acid sequence.
  • natural occurring proteins may be produced by culturing the organism expressing the protein and subsequently purifying the protein or it may be produced by cloning a nucleic acid, e.g. genomic DNA or cDNA, encoding the protein into an expression vector, introducing said expression vector into a host cell, culturing the host cell and purifying the expressed protein.
  • protein variants may be produced by site-directed mutagenesis of a parent protein, introduction into expression vector, host cell etc.
  • the parent protein may be cloned from a strain producing the polypeptide or from an expression library, i.e. it may be isolated from genomic DNA or prepared from cDNA, or a combination thereof.
  • subtilase or subtilase or subtilase variant of the invention.
  • suitable techniques reference is made to Molecular cloning: A laboratory manual (Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.)); Current protocols in Molecular Biology (John Wiley and Sons, 1995; Harwood, C. R., and Cut- ting, S. M.
  • Random mutagenesis is suitably performed either as localized or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid se- quence shown in question, or within the whole gene.
  • the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions that are to be changed.
  • the doping or spiking may be done so that codons for unwanted amino acids are avoided.
  • the doped or spiked oligonucleotide can be incorporated into the DNA encoding the subtilase enzyme by any published technique, using, e.g., PCR, LCR or any DNA polymerase and ligase as deemed appropriate.
  • the doping is carried out using "constant random doping", in which the percentage of wild-type and modification in each position is predefined.
  • the doping may be directed toward a preference for the introduction of certain nucleotides, and thereby a preference for the introduction of one or more specific amino acid residues.
  • the doping may be made, e.g., so as to allow for the introduction of 90% wild type and 10% modifications in each position.
  • An additional consideration in the choice of a doping scheme is based on genetic as well as protein-structural constraints.
  • the doping scheme may be made by using the DOPE program which, inter alia, ensures that introduction of stop codons is avoided (L.J. Jensen et al. Nucleic Acid Research, 26, 697-702 (1998).
  • PCR-generated mutagenesis When PCR-generated mutagenesis is used, either a chemically treated or non- treated gene encoding a parent subtilase enzyme is subjected to PCR under conditions that increase the misincorporation of nucleotides (Deshler 1992; Leung et al., Technique, 1 , 1989, pp. 11-15).
  • the DNA sequence to be mutagenized may conveniently be present in a genomic or cDNA library prepared from an organism expressing the parent subtilase.
  • the DNA sequence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or otherwise exposed to the mutagenising agent.
  • the DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harbored in the cell. Fi- nally, the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence.
  • telomere amplification may be performed in accordance with methods known in the art, the presently preferred method being PCR-generated amplification using oligonucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme.
  • the mutated DNA sequence may further comprise a DNA sequence encoding func- tions permitting expression of the mutated DNA sequence.
  • the random mutagenesis may be advantageously localised to a part of the parent subtilase i n q uestion. This may, e .g., be advantageous when certain regions of the e n- zyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.
  • the localised or region-specific, random mutagenesis is conveniently performed by use of PCR generated mutagenesis techniques as described above or any other suitable technique known in the art.
  • the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g., by insertion into a suitable vector, and said part m ay be subsequently subjected to m utagenesis by use of any of the m utagenesis methods discussed above.
  • the random mutagenesis may be carried out by the following steps:
  • Suitable dope algorithms for use in step 6 are well known in the art.
  • One such algorithm is described by Tomandl, D. et al., 1997, Journal of Computer-Aided Molecular Design 11 :29-38.
  • Another algorithm is DOPE (Jensen, LJ, Andersen, KV, Svendsen, A, and o Kretzschmar, T (1998) Nucleic Acids Research 26:697-702).
  • a recombinant expression vector comprising a nucleic acid sequence encoding a subtilase variant of the invention may be any vector that may conveniently be subjected to 5 recombinant DNA p rocedures a nd which may bring about the expression of the nucleic acid sequence.
  • the choice of vector will often depend on the host cell into which it is to be intro- prised.
  • a suitable vector include a linear or closed circular plasmid or a virus.
  • the vector may be an autonomously replicating vector, i.e., a vector which exists as an 0 extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini chromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, pACYC184, pUB110, pE194, pTA1060, and pAM ⁇ l .
  • Examples of origin of 5 replications for use in a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARS1.
  • the origin of replication may be one having a mutation which makes it function as temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • Vectors which are integrated into the genome of the host cell may contain any nucleic acid sequence enabling integration into the genome, in particular it may contain nucleic acid sequences facilitating integration into the genome by homolo- 5 gous or non-homologous recombination.
  • the vector system may be a single vector, e.g. plasmid or virus, or two or more vectors, e.g. plasmids or virus', which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the vector may in particular be an expression vector in which the DNA sequence encoding the subtilase variant of the invention is operably linked to additional segments or control sequences required for transcription of the DNA.
  • the term, "operably linked" indi- cates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence encoding the subtilase variant.
  • Additional segments or control sequences include a promoter, a leader, a polyadenylation sequence, a propeptide sequence, a signal sequence and a transcription terminator. At a minimum the control sequences include a pro- moter and transcriptional and translational stop signals.
  • the promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for use in bacterial host cells include the promoter of the Bacillus subtilis levansucrase gene (sacB), the Bacillus stearothermophilus malto- genic amylase gene (amyM), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, o
  • promoters for use in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhi- zomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae those phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (as described in U.S.
  • Patent No. 4,288,627 which is incorporated herein by reference
  • Particularly preferred promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes en- coding Aspergillus niger neutral (-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters.
  • Further suitable promoters for use in filamentous fungus host cells are the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093 - 2099) or the tpiA promoter.
  • promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419 - 434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPM (US 4,599,311 ) or ADH2-4c (Russell et al., Nature 304 (1983), 652 - 654) promoters.
  • yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.
  • useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).
  • SV40 Simian Virus 40
  • RSV Rous sarcoma virus
  • BPV bovine papilloma virus
  • Suitable promoters for use in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981 ), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814) or the adenovirus 2 major late promoter.
  • a suitable promoter for use in insect cells is the polyhedrin promoter (US 4,745,051 ; Vasuvedan et al., FEBS Lett. 311 , (1992) 7 - 11), the P10 promoter (J.M. Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica polyhedro- sis virus basic protein promoter (EP 397 485), the baculovirus immediate early gene 1 promoter (US 5,155,037; US 5,162,222), or the baculovirus 39K delayed-early gene promoter (US 5,155,037; US 5,162,222).
  • the polyhedrin promoter US 4,745,051 ; Vasuvedan et al., FEBS Lett. 311 , (1992) 7 - 11
  • the P10 promoter J.M. Vlak et al., J. Gen. Virology 69, 1988, pp
  • the DNA sequence encoding a subtilase variant of the invention may also, if necessary, be operably connected to a suitable terminator.
  • the recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like ampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, neomycin, hygromycin, methotrexate, or resistance to heavy metals, virus or herbicides, or which provides for prototrophy or auxotrophs.
  • bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, resistance.
  • a frequently used mammalian marker is the dihydrofolate reductase gene (DHFR).
  • Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3.
  • a selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate d ecarboxylase), sC ( sulfate a denyltransferase), trpC (anthranilate synthase), and g lufosinate resistance markers, as well as equivalents from other species.
  • amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus.
  • selection may be accomplished by co-transformation, e.g., as described in WO 91/17243, where the selectable marker is on a separate vector.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector.
  • the secretory signal sequence is joined to the DNA sequence encoding the enzyme in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5 * to the DNA sequence encoding the enzyme.
  • the secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein.
  • More than one copy of a nucleic acid sequence encoding an enzyme of the present invention may be inserted into the host cell to amplify expression of the nucleic acid sequence.
  • Stable amplification of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome using methods well known in the art and selecting for transformants.
  • the nucleic acid constructs of the present invention may also comprise one or more nucleic acid sequences which encode one or more factors that are advantageous in the expression of the polypeptide, e.g., an activator (e.g., a trans-acting factor), a chaperone, and a processing protease. Any factor that is functional in the host cell of choice may be used in the present invention.
  • an activator e.g., a trans-acting factor
  • a chaperone e.g., a chaperone
  • processing protease e.g., a factor that is functional in the host cell of choice.
  • the nucleic acids encoding one or more of these factors are not necessarily in tandem with the nucleic acid sequence encoding the polypeptide.
  • the DNA sequence encoding a subtilase variant of the present invention may be either homologous or heterologous to the host cell into which it is introduced. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal se- quence and/or terminator sequence than in its natural environment.
  • the term "homologous” is intended to include a DNA sequence encoding an enzyme native to the host organism in question.
  • heterologous is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence.
  • the host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell that is capable of producing the present subtilase variants, such as prokaryotes, e.g. bacteria or eukaryotes, such as fungal cells, e.g. yeasts or filamentous fungi, insect cells, plant cells or mammalian cells.
  • prokaryotes e.g. bacteria
  • eukaryotes such as fungal cells, e.g. yeasts or filamentous fungi, insect cells, plant cells or mammalian cells.
  • Examples of bacterial host cells which, on cultivation, are capable of producing the subtilase variants of the invention are gram-positive bacteria such as strains of Bacillus, e.g. strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gram- negative bacteria such as Escherichia coli or Pseudomonas sp.
  • the transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
  • the enzyme When expressing the subtilase variant in bacteria such as E. coli, the enzyme may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or it may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the enzyme is refolded by diluting the denaturing agent. In the latter case, the enzyme may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the enzyme.
  • gram-positive bacteria such as Bacillus or
  • the enzyme may be retained in the cytoplasm, or it may be directed to the extracellular medium by a bacterial secretion sequence. In the latter case, the enzyme may be recovered from the medium as described below.
  • yeast cells include cells of a species of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, Hansehula, or Yarrowia.
  • the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cer- evisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell.
  • yeast host cells are a Kluyveromyces lactis, Kluyveromyces fragilis, Hansehula polymorpha, Pichia pastoris, Yarrowia lipolytica, Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose, Pichia guillermondii and Pichia methanolio cell (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; US 4,882,279 and US 4,879,231).
  • yeast Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980.
  • yeast and manipulation of yeast genetics are well known in the art (see, e.g., Biochemistry and Genetics of Yeast, Bacil, M.,MHorecker, B.J., and Stopani, A.O.M., editors, 2nd edition, 1987; The Yeasts, Rose, A.H., and Harrison, J.S., editors, 2nd edition, 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathern et al., editors, 1981). Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N.
  • filamentous fungal cells include filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra), in particular it may of the a cell of a species of Acremonium, such as A. chrysogenum, Aspergillus, such as A. awamori, A. foetidus, A. japonicus, A. niger, A. nidulans or A. oryzae, Fusarium, such as F. bactridioides, F. cerealis, F. crookwellense, F. culmorum, F. graminearum, F. graminum, F. heterosporum, F. negundi, F.
  • a species of Acremonium such as A. chrysogenum, Aspergillus, such as A. awamori, A. foetidus, A. japonicus, A. niger, A. nidulans or A.
  • reticulatum F. roseum, F. sambucinum, F. sarcochroum, F. sulphureum, F. trichothecioides or F. oxysporum, Humicola, such as H. insolens or H. lanuginose, Mucor, such as M. miehei, Myceliophthora, such as M. thermo- philum, Neurospora, such as N. crassa, Penicillium, such as P. purpurogenum, Thielavia, such as T. terrestris, Tolypocladium, or Trichoderma, such as T. harzianum, T. koningii, T. longibrachiatum, T. reesei or T. viride, or a teleomorph or synonym thereof.
  • Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 230 023.
  • insect cells include a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. US 5,077,214). Culture conditions may suitably be as described in WO 89/01029 or WO 89/01028.Transformation of insect cells and pro- duction of heterologous polypeptides therein may be performed as described in US 4,745,051 ; US 4, 775, 624; US 4,879,236; US 5,155,037; US 5,162,222; EP 397,485).
  • mammalian cells examples include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection. Methods of transfect- ing mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601 - 621 ; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327 - 341 ; Loyter et al., Proc. Natl. Acad. Sci.
  • Mammalian cells may be transfected by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52:546).
  • the above mentioned host cells transformed or transfected with a vector comprising a nucleic acid sequence encoding an enzyme of the present invention are typically cultured in a suitable nutrient medium under conditions permitting the production of the desired molecules, after which these are recovered from the cells, or the culture broth.
  • the medium used to culture the host cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The media may be prepared using procedures known in the art (see, e.g., references o for bacteria and yeast; Bennett, J.W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).
  • the enzymes of the present invention are secreted into the nutrient medium, they may be recovered directly from the medium. If they are not secreted, they may be recovered from cell lysates.
  • the enzymes of the present invention may be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the enzyme in question.
  • the enzymes of the invention may be detected using methods known in the art that are specific for these proteins. These detection methods include use of specific antibodies, formation of a product, or disappearance of a substrate. For example, an enzyme assay may be used to determine the activity of the molecule. Procedures for determining various kinds of activity are known in the art.
  • the enzymes of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J-C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction
  • IEF isoelectric focusing
  • differential solubility e.g., ammonium sulfate precipitation
  • extraction see, e.g
  • heterologous host cell An advantage of using a heterologous host cell is that it is possible to make a highly purified enzyme composition, characterized in being free from homologous impurities, which are often present when a protein or peptide is expressed in a homologous host cell.
  • homologous impurities mean any impurity (e.g. other polypeptides than the enzyme of the invention) which originates from the homologous cell where the enzyme of the invention is originally obtained from.
  • the enzyme of the invention may be added to and thus become a component of a detergent composition.
  • the d etergent composition of the i nvention m ay for example be formulated a s a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations, especially for automatic dish washing (ADW).
  • a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations, especially for automatic dish washing (ADW).
  • ADW automatic dish washing
  • the invention provides a detergent additive comprising the en- zyme of the invention.
  • the detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galac- tanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.
  • enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galac- tanase, a
  • Suitable proteases include those of animal, vegetable or microbial origin. Mi- crobial origin is preferred. Chemically modified or protein engineered mutants are included.
  • the protease may be a serine protease or a metallo protease, preferably an alkaline mi- crobial protease or a trypsin-like protease.
  • alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carisberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
  • trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
  • useful proteases are the variants described in WO 92/19729, WO
  • Preferred commercially available protease enzymes include AlcalaseTM, Savi- naseTM, PrimaseTM, DuralaseTM, EsperaseTM, and KannaseTM (Novozymes A/S), MaxataseTM, MaxacalTM, MaxapemTM, ProperaseTM, PurafectTM, Purafect OxPTM, FN2TM, and FN3TM (Genencor International Inc.).
  • Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseu- domonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. ce- pacia (EP 331 376), P. stutzeri (GB 1 ,372,034), P.
  • lipase variants such as those described in WO 92/05249, WO 94/01541 , EP 407 225, EP 260 1 05, WO 95/35381 , WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.
  • LipolaseTM and Lipolase UltraTM are preferred commercially available lipase enzymes.
  • Amylases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, ⁇ -amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, de- scribed in more detail in GB 1 ,296,839.
  • Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391 , 408, and 444.
  • amylases are DuramylTM, TermamylTM, FungamylTM and BANTM (Novozymes A/S), RapidaseTM and PurastarTM (from Genencor International Inc.).
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modi- fied or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259. Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits.
  • cellulases examples include cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940.
  • cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299.
  • cellulases include CelluzymeTM, and CarezymeTM (Novozymes A/S), ClazinaseTM, and Puradax HATM (Genencor International Inc.), and KAC- 500(B)TM (Kao Corporation).
  • Peroxidases/Oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include GuardzymeTM (Novozymes A/S).
  • the detergent enzyme(s) may be included i n a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes.
  • a detergent additive of the invention i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc.
  • Preferred detergent additive formulations are granulates, in particular non-dusting granu- lates, liquids, in particular stabilized liquids, or slurries.
  • Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 30000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
  • Pro- tected enzymes may be prepared according to the method disclosed in EP 238,216.
  • the detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid.
  • a liquid detergent may be aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or non-aqueous.
  • the detergent composition comprises one or more surfactants, which may be non- ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic.
  • the surfactants are typically present at a level of from 0.1% to 60% by weight.
  • the detergent When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, al- pha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
  • an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, al- pha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
  • the detergent When included therein the detergent will usually contain from about 0.2% to about
  • glucamides N-acyl N-alkyl derivatives of glucosamine
  • the detergent may contain 0-65 % of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic a cid, d iethylenetriaminepentaacetic a cid, a Ikyl- o r a Ikenylsuc- cinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic a cid, d iethylenetriaminepentaacetic a cid, a Ikyl- o r a Ikenylsuc- cinic acid, soluble silicates or layered silicates
  • the detergent may comprise one or more polymers.
  • examples are carboxymethyl- cellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylat.es such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • the detergent may contain a bleaching system which may comprise a H 2 0 2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
  • a bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.
  • the enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.
  • the detergent may also contain other conventional detergent ingredients such as e.g.
  • fabric conditioners i ncluding clays, foam boosters, suds s uppressors, a nti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • any enzyme in particular the enzyme of the inven- tion, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per litre of wash liquor, preferably 0.05-5 mg of enzyme protein per litre of wash liquor, in particular 0.1-1 mg of enzyme protein per litre of wash liquor.
  • the enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.
  • Standard textile pieces are obtained from EMPA St. Gallen, Lerchfeldstrasse 5, CH-9014 St. Gallen, Switzerland.
  • type EMPA 116 cotton textile stained with blood, milk and ink
  • EMPA 117 polyyester/cotton textile stained with blood, milk and ink
  • Other atandard textile pieces are obtained from wfk-Cleaning Technology Research Institute, Chris- tenfeld 10, D-41379 Br ⁇ ggen-Bracht, Germany.
  • type wfk10N cotton textile stained with egg/pigment
  • wfklOeggEG cotton textile stained with egg yolk. Denaturation of wfk10N occurs in an autoclave.
  • the present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
  • a suitable host cell which has been transformed with a DNA sequence encoding the enzyme
  • the resulting enzyme is recovered from the culture.
  • an expression vector comprising a DNA sequence encoding the enzyme is transformed into a heterologous host cell it is possible to enable heterologous recombinant production of the enzyme of the invention.
  • the medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question.
  • the expressed subtilase may conveniently be secreted into the culture medium and may be recovered there-from by well- known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • Subtilase JP170 site-directed variants of the invention comprising specific inser- tions/deletions/substitutions are made by traditional cloning of DNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) produced by PCR with oligos containing the desired mutations.
  • the template plasmid DNA may be pSX222, or an analogue of this containing a variant of subtilisin JP170. Mutations are introduced by oligo directed mutagenesis to the construction of variants.
  • subtilisin JP170 variants were transformed into E. coli. DNA purified from an over night culture of these transformants is transformed into B. subtilis by restriction endonuclease digestion, purification of DNA fragments, ligation, transformation of S. subtilis. Transformation of B. subtilis i s performed as described by Dubnau et al., 1971 , J. M ol. Biol. 56, pp. 209-221.
  • Site-directed mutagenesis in order to introduce mutations in a specific region The overall strategy used to perform site-directed mutagenesis is: Mutagenic primers (oligonucleotides) are synthesized corresponding to the DNA sequence flanking the sites of mutation, separated by the DNA base pairs defining the insertions / deletions / substitutions.
  • the resulting mutagenic primers are used in a PCR reaction with the modified plasmid pSX222.
  • the resulting PCR fragment is purified and extended in a sec- ond PCR-reaction, the resulting PCR product is purified and extended in a third PCR- reaction before being digegted by endonucleases and cloned into the E. coli - B. subtilis shuttle vector pSX222.
  • the PCR reactions are performed under normal conditions.
  • the plasmid DNA is transformed into E. coli by well-known techniques and one E. coli colony is sequenced to confirm the mutation designed.
  • the pSX222 expression plasmid comprising a variant of the invention was transformed into a competent B. subtilis strain and fermented as described above.
  • subtilisin variants After fermentation, purification of subtilisin variants is accomplished u sing H ydro- phobic Charge Induction Chromatography (HCIC) and subsequent vacuum filtration.
  • the HCIC uses a cellulose matrix to which 4-Mercapto-Ethyl- Pyridine (4-MEP) is bound. Beads of the cellulose matrix sized 80-100 ⁇ m are mixed with a media containing yeast and the transformed B. subtilis capable of secreting the subtilisin variants and incu- bated at pH 9.5 in Unifilter ® microplates.
  • the pH is now lowered by washing the filter with a n elution b uffer ( pH 5 ) H ereby the e nzymes part from the beads a nd can be retrieved from the buffer.
  • the concentration of the purified subtilisin enzyme variants is assessed by active site titration (AST).
  • the purified enzyme is incubated with the high affinity inhibitor CI-2A at different concentrations to inhibit a varying amount of the active sites.
  • the protease and inhibitor o binds to each other at a 1 :1 ratio and accordingly the enzyme concentration can be directly related to the concentration of inhibitor, at which all protease is inactive.
  • a substrate 0.6 mM Suc-Ala-Ala-Pro-Phe-pNA in Tris/HCI buffer
  • pNA paranitrophenol
  • the performance of the enzyme variant is measured as the brightness of the colour of the textile samples washed with that specific protease. Brightness can also be expressed as the intensity of the light reflected from the textile sample when illuminated with white light. When the textile is stained the intensity of the reflected light is lower, than that of a c lean t extile. T herefore t he i ntensity o f t he r eflected I ight c an be u sed t o m easure wash performance of a shuffled protease.
  • Colour measurements are made with a professional flatbed scanner (PFU DL2400pro, obtainable from: J.M. Thomsen, Dorfgade 2, Dorf, Dronninglund, DK-9330), which is used to capture an image of the washed textile samples.
  • the scans are made with a resolution of 200 dpi and with an output colour dept of 24 bits.
  • the scanner is frequently calibrated with a Kodak reflective IT8 target.
  • Detergents for wash performance tests of the subtilases of the invention can be ob- tained by purchasing fully formulated commercial detergents at the market and subsequently inactivate the enzymatic components by heat treatment (5 minutes at 85°C in aqueous solution).
  • a commercial detergent base without enzymes can be purchased directly from the manufacturer.
  • a suitable model detergent can be purchased and used for wash performance tests.
  • the proteases may also be tested in a model detergent composition comprising
  • subtilases according to the invention e xhibits i mproved wash p erformance o n e gg s tains i n c omparison t o t he wiild type JP170 subtilase with SEQ ID NO:1.
  • REMARK Complex of JP170 and CI2A inhibitor REMARK Contents of asymmetric unit subtilisin 2x (433 a. a. x 2) REMARK CI2A inhibitor 2x (a. a. 16 - 83 and 21 - 83) REMARK small peptide (autodigestion product, a. a.
  • REMARK 3 METHOD USED BABINET MODEL WITH MASK REMARK 3 PARAMETERS FOR MASK CALCULATION REMARK 3 VDW PROBE RADIUS 1.40 REMARK 3 ION PROBE RADIUS 0.80 REMARK 3 SHRINKAGE RADIUS 0.80 REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: REMARK 3 HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS REMARK 3 CISPEP 1 GLY A 163 PRO A 164 00 CISPEP 2 ALA A 171 PRO A 172 00 CISPEP 3 PHE A 191 GLY A 192 00 CISPEP 4 ASN A 199 HIS A 200 00 CISPEP 5 GLY A 208 PRO A 209 00 CISPEP 6 LYS A 216 PRO A 217 00 CISPEP 7 ASP A 236 SER A 237 00 CISPEP 8 ASP A 244 SER A 245 00 CISPEP 9
  • HETATM 3 O10 ASN A 1 19.168 22 .251 -2.661 1.00 13 .33 A O
  • ATOM 106 CA ASN A 15 30 .185 10 .847 3. .469 1, .00 20 .71 A C
  • ATOM 180 C GLN A 24 13 .619 2 .022 2, .352 1 .00 13 .19 A C
  • ATOM 182 N ILE A 25 12 .324 2 .066 2, .692 1 .00 13 .28 A N
  • ATOM 220 C ASP A 30 0, .665 13 .113 -5. .327 1, .00 12 .41 A C
  • ATOM 266 CZ ARG A 37 4, .322 5, .434 -20, .575 1, .00 38, .63 A C
  • ATOM 272 CA ASN A 38 7 .254 1. .998 -13, .703 1, .00 20 .37 A C
  • ATOM 308 CA HIS A 43 12, .479 9 .903 -15, .081 1, .00 17, .38 A C
  • ATOM 326 N ALA A 45 15, .665 9 .954 -12, .152 1, .00 16 .39 A N
  • ATOM 373 O ILE A 50 9 .339 -0 .161 -7, .909 1, .00 18 .69 A O
  • ATOM 382 CA ALA A 52 5, .179 0 .338 -6, .052 1, .00 17 .34 A C
  • ATOM 407 CA ALA A 55 -3 .032 5 .260 -8. .926 1 .00 17 .49 A C
  • ATOM 410 O ALA A 55 -5 .174 4 .400 -8. .269 1 .00 19 .60 A O
  • ATOM 429 CZ ARG A 58 -3. ,248 11, .716 -12. ,392 1. ,00 18. .78 A C
  • ATOM 442 CA ASN A 60 -4, .939 3 .618 -14, ,124 1, .00 21 .57 A C
  • ATOM 458 CA ALA A 62 -0, .418 7 .910 -11, .902 1, .00 16 .34 A C
  • ATOM 461 O ALA A 62 1 .583 9 .221 -11, .728 1, .00 15 .61 A O
  • ATOM 472 CB ASP A 64 3 .450 13 .990 -12, .233 1 .00 14 .59 A C
  • ATOM 478 N PRO A 65 1 .065 15 .644 -14 .476 1 .00 16 .18 A N
  • ATOM 479 CA PRO A 65 0 .950 16 .813 -15 .343 1 .00 17 .33 A C
  • ATOM 482 CD PRO A 65 -0 .249 15 .043 -14 .172 1 .00 17 .21 A
  • C ATOM 483 C PRO A 65 1.228 18.102 -14.607 1.00 17.72 A C

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Detergent Compositions (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The present invention relates to methods for producing variants of a parent JP170 subtitlase and of a parent BPN' subtilase and to JP170 and BPN' variants having altered proper-ties as compared to the parent JP170/BPN' subtilase.

Description

SUBTILASES
FIELD OF THE INVENTION
The present invention relates to JP170 and BPN' like subtilases and to methods of construction such variants with altered properties, such as stability (e.g. thermostability or storage stability), Ca2+ dependency, pH dependent activity, improved performance in washing and cleaning applications.
BACKGROUND OF THE INVENTION
Enzymes have been used within the detergent industry as part of washing formula- tions for more than 30 years. Proteases are from a commercial perspective the most relevant enzyme in such formulations, but other enzymes including lipases, amylases, cellu- lases or mixtures of enzymes are also often used.
To improve the cost and/or the performance of proteases there is an ongoing search for proteases w ith altered properties, such as i ncreased activity at low tempera- tures, increased thermostability, increased specific activity at a given pH, altered Ca2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc.
The search for proteases with altered properties include both discovery of naturally occurring proteases, i.e. so called wild-type proteases but also alteration of well-known proteases by e.g. genetic manipulation of the nucleic acid sequence encoding said proteases. Knowledge of the relationship between the three-dimensional structure and the function of a protein has improved the ability to evaluate which areas of a protein to alter to affect a specific characteristic of the protein.
One family of p roteases, which are often u sed i n d etergents, a re the subtilases. This family has previously been further grouped into 6 different sub-groups by Siezen RJ and Leunissen JAM, 1997, Protein Science, 6, 501-523. One of these sub-groups is the Subtilisin family which includes subtilases such as BPN', subtilisin 309 (SAVINASE®, NOVOZYMES A/S), subtilisin Carlsberg (ALCALASE®, NOVOZYMES A/S), subtilisin S41 (a subtilase from the psychrophilic Antarctic Bacillus TA41 , Davail S et al. 1994, The Journal of B iological Chemistry, 269(26), 99. 1 7448-17453), s ubtilisin S 39 (a s ubtilase from the psychrophilic Antarctic Bacillus TA39, Narinx E et al. 1997, Protein Engineering, 10 (11 ), pp. 1271-1279) and TY145 (a subtilase from Bacillus sp. TY145, NCIMB 40339 described in WO 92/17577).
The groupings indicated above were made based on primary sequence alignments, with only little consideration of three-dimensional structure. However, despite sequence homologies between subtilases belonging to the Subtilisin subgroup, modelling of the three-dimensional structure of one subtilase on the basis of the three-dimensional structure of another subtilase (such as the subtilisin BPN' that was used by Siezen and Leunis- sen) may result in an incorrect three-dimensional model structure because of structural differences. Recently the three-dimensional structure of subtilase TY145 have been elucidated and it was found that there are several differences between this and the three-dimensional structure of BPN' also belonging to the Subtilisin subgroup of subtilases (PCT/DK2004/000066).
The differences between the three-dimensional structures of TY145 and BPN' are confirmed b y t he t hree-dimensional s tructure of the s ubtilase " sphericase" f rom Bacillus sphaericus (PDB NO:1 EA7, Protein Data Bank). The overall structure and many details of this subtilase are very homologous with the TY145 subtilase structure.
The subtilase JP170 and subtilases similar to JP170 are already known in the art, but the three-dimensional structure has not been disclosed for such subtilases. The JP170 subtilase was described as protease A in WO 88/01293 to Novozymes.
Later the patent application WO 98/56927 to Novozymes Biotech disclosed the amino acid (polypeptide) sequence of JP170 and the DNA sequence encoding JP170. In EP 204 342 the protease Ya was disclosed, and JP7-62152 and JP 4197182 to Lion Corp. disclosed the DNA sequence encoding protease Ya produced by Bacillus sp. Y that is homologous to JP170. In addition US 6,376,227 to Kao Corp. discloses physical characteristics as well as DNA and polypeptide sequences of alkaline proteases KP43, K P1790 and KP9860 which are also homologous to JP170. Recently variants of the KP43, KP9860, SD-521 and Ya proteases among others were disclosed in EP 1209233. These proteases are highly homologous, and an alignment of KP43, KP9860, SD-521 , Y and JP170 revealed at least 90% homology. Therefore JP170, Ya and SD-521 represent these proteases in the alignment of Fig. 1 of the present specification.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows an alignment of three JP170 type proteases: (a) SD-521 (EP 1 209 233), (b) protease Y a (WO 99/67370), and (c) JP170 (WO 98/56927, mature sequence from Appendix 1 ).
Figure 2 shows a superposition of the 3D structures of the proteases JP170 and Savinase ( BLSAVI), with i ndication of calcium b inding s ites. I n the figure J P170 i s i ndicated in light grey with three ion-binding sites, and Savinase in a dark structure with two ion-binding sites.
Figure 3 shows a matrix of homology between amino acid sequences of subtilases pertaining to various subtilase subgroups. The sequences are identified by sequence database accession numbers and their derivation. 1 : aam50084; Subtilase derived from Bacillus sp. strain SD-521 2: aaw89547; Subtilase derived from Bacillus sp. JP170 3: q45681 ; Subtilase derived from B. subtilis (BSTA41 )
4: p28842; Psychrophilic subtilisin derived from Antarctic Bacillus strain (BSTA39) 5: abb77095; Subtilase derived from Bacillus sp. (TY145)
6: p00783; Subtilase derived from Bacillus subtilis var. amylosacchariticus (BSAMY) 7: p29142; Subtilase derived from Bacillus stearothermophilus (BSSJ) 8: p35835; Subtilase derived from Bacillus subtilis var. natto. (BSNAT)
9: p07518; Subtilase derived from Bacillus pumilus (B. mesentericus) (BPMES) 10: p00782; Subtilase derived from Bacillus amyloliquefaciens (BPN') 11 : p00780; Subtilase derived from Bacillus licheniformis (BLSCAR) 12: p41363; Subtilase derived from Bacillus halodurans (BHSAH) 13: aaw62222; Subtilase derived from Bacillus lentus (BLS147)
14: p29600; Subtilase derived from Bacillus lentus (BLSAVI, BLS309) 15: p27693; Subtilase derived from Bacillus alcalophilus (BAALKP) 16: q99405; Subtilase derived from Bacillus sp. strain KSM-K16 (BSKSMK) 17: p29599; Subtilase derived from Bacillus lentus (BLSUBL). Sequences 1 and 2 belong to the JP170 type, sequences 3 to 5 belong to the
TY145 type, sequences 6 to 11 belong to the "true subtilisins" or I-S1 type, and sequences 12 to 17 belong to the "highly alkaline" subtilisins or I-S2 type. From Fig. 3 it is clear that these types are quite distinct.
Figure 4 shows a three-dimensional alignment of the subtilases: (1 ) Ty145; (2) BPN'; (3) Savinase; and (4) JP170.
By 3D sequences is meant that the position of homologous residues are chosen by superposition of the 3D structures and subsequently the amino acid sequences are aligned based on these homologous positions.
BRIEF DESCRIPTION OF THE APPENDIX
APPENDIX 1 s hows the structural coordinates for the solved crystal structure of JP170.
SEQUENCE LISTING In the appended sequence listing the following amino acid sequences are provided:
Subtilase JP170 (SEQ ID NO:1) Subtilase Y (SEQ ID NO:2) Subtilase SD-521 (SEQ ID NO:3) Subtilase BPN" (SEQ ID NO:4) Partiel sequence (SEQ ID NO:5) Partiel sequence (SEQ ID NO:6) Subtilase TY145 (SEQ ID NO:7)
BRIEF DESCRIPTION OF THE INVENTION
Now the inventors of the present invention disclose the three-dimensional structure of the subtilase JP170. This subtilase has large structural differences to the structures of the subtilisins BPN' and TY145.
Based on these differences the inventors have modified the amino acid sequence of subtilases having a JP170 type structure and subtilases having a BPN' type structure to obtain variants with improved properties. The variants have altered properties, such as in- creased activity at low temperatures, increased thermostability, increased specific activity at a given pH, altered Ca2+ dependency, increased stability in the presence of other detergent ingredients (e.g. bleach, surfactants etc.) etc.
Accordingly, the object of the present invention is to provide a method for constructing subtilases having altered properties, in particular to provide a method for constructing subtilases having altered properties as described above.
Thus the present invention relates to a method for constructing a variant of a parent subtilase, wherein the variant has at least one altered property as compared to said parent subtilase, which method comprises: a) analyzing the three-dimensional structure of the subtilase to identify, on the basis of an evaluation of structural considerations in relation to a JP170 three dimensional structure, at least one amino acid residue or at least one structural region of the subtilase, which is of relevance for altering said property; b) modifying the DNA of the polynucleotide encoding the parent to construct a polynucleotide encoding a variant subtilase, which in comparison to the parent sub- tilase, has been modified by deletion, substitution or insertion of the amino acid residue or structural part identified in i) so as to alter said property; c) expressing the variant subtilase in a suitable host, and d) testing the resulting subtilase variant for said property.
More specifically the invention relates to a method of producing a subtilase variant, wherein the variant has at least one altered property as compared to a parent subtilase, which method comprises: a) producing a model structure of the parent subtilase on the three-dimensional structure of BPN', TY145 or JP170; or producing an actually determined three- dimensional structure of the parent subtilase, b) comparing the model or actual three-dimensional structure of the parent subtilase to the JP170 structure by superimposing the structures through matching the CA, CB, C, O, and N atoms of the active site residues, c) identifying on the basis of the comparison in step b) at least one structural part of the parent subtilase, wherein an alteration in said structural part is predicted to re- suit in an altered property; d) modifying the n ucleic a cid sequence e ncoding the p arent subtilase to p roduce a nucleic acid sequence encoding at least one deletion or substitution of one or more amino acids at a position corresponding to said structural part, or at least one insertion of one or more amino acid residues in positions corresponding to said struc- tural part; e) performing steps c) and d) iteratively N times, where N is an integer with the value of one or more; f) preparing the variant resulting from steps a) - e); g) testing the properties of said variant; and h) optionally repeating steps a) - g) recursively; and i) selecting a subtilase variant having at least one altered property as compared to the parent subtilase. j) expressing the modified nucleic acid sequence in a host cell to produce the variant subtilase; k) isolating the produced subtilase variant;
I) purifying the isolated subtilase variant and m) recovering the purified subtilase variant.
Although it has been described in the following that modification of the parent subti- lase in certain regions and/or positions is expected to confer a particular effect to the thus produced subtilase variant, it should be noted that modification of the parent subtilase in any of such regions may also give rise to any other of the above-mentioned effects. For example, any of the regions and/or positions mentioned as being of particular interest with respect to, e.g., improved thermostability, may also give rise to, e.g., higher activity at a lower pH, an altered pH optimum, or increased specific activity, such as increased pepti- dase activity. Further aspects of the present invention relates to variants of a subtilase, t_he DNA encoding such variants and methods of preparing the variants. Still further aspect s of the present invention relates to the use of the variants for various industrial purposes, in particular as an additive in detergent compositions. Other aspects of the present invent tion will be apparent from the below description as well as from the appended claims.
DEFINITIONS
Prior to discussing this invention in further detail, the following terms and conventions will first be defined. For a detailed description of the nomenclature of amino acids and nucleic acids, we refer to WO 00/71691 page 5, hereby incorporated by reference. A description of the nomenclature of modifications i ntroduced i n a polypeptide by g enetic m anipulation can be found in WO 00/71691 page 7-12, hereby incorporated by reference.
The term "subtilases" refer to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serine proteases or serine peptidases is a subgroup of proteases characterised by having a serine in the active site, which forms a covalent adduct with the substrate. Further the subtilases (and the serine proteases) are characterised by having two active site amino acid residues apart from the serine, namely a histidine and an aspartic acid residue. Subtilases are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. The subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family. The Subtilisin family (EC 3.4.21.62) may be further divided into 3 sub-groups, i.e. I-
S1 ("true" subtilisins), I-S2 (highly alkaline proteases) and intracellular subtilisins. Definitions or grouping of enzymes may vary or change, however, in the context of the present invention the above division of subtilases into sub-division or sub-groups shall be understood as those described by Siezen et al., Protein Engng. 4 (1991 ) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523.
The term "parent" is in the context of the present invention to be understood as a protein, which is modified to create a protein variant. The parent protein may be a naturally occurring (wild-type) polypeptide or it may be a variant thereof prepared by any suitable means. For instance, the parent protein may be a variant of a naturally occurring protein which has been modified by substitution, chemical modification, deletion or truncation of one or more amino acid residues, or by addition or insertion of one or more amino acid residues to the amino acid sequence, of a naturally-occurring polypeptide. Thus the term "parent subtilase" refers to a subtilase which is modified to create a subtilase variant.
The term "variant" is in the context of the present invention to be understood as a protein which has been modified as compared to a parent protein at one or more amino acid residues.
The term "modification(s)" or "modified" is in the context of the present invention to be understood as to include chemical modification of a protein as well as genetic manipulation of the DNA encoding a protein. The modification(s) may be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest. Thus the term "modified protein", e.g. "modified subtilase", is to be understood a s a protein which contains modification(s) compared to a parent p rotein, e.g. subtilase.
"Homology" or "homologous to" is in the context of the present invention to be understood in its conventional meaning and the "homology" between two amino acid se- quences should be determined by use of the "Similarity" defined by the GAP program from the University of Wisconsin Genetics Computer Group (UWGCG) package using default settings for alignment parameters, comparison matrix, gap and gap extension penalties. Default values for GAP penalties, i.e. GAP creation penalty of 3.0 and GAP extension penalty of 0.1 (Program Manual for the Wisconsin Package, Version 8, August 1994, Ge- netics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 ). The method is also described in S.B. Needleman and CD. Wunsch, Journal of Molecular Biology, 48, 443-445 (1970). Identities can be extracted from the same calculation. The homology between two amino acid sequences can also be determined by "identity" or "similarity" using the GAP routine of the UWGCG package version 9.1 with default setting for alignment parameters, comparison matrix, gap and gap extension penalties can also be applied using the following parameters: gap creation penalty = 8 and gap extension penalty = 8 and all other parameters kept at their default values. The output from the routine is besides the amino acid alignment the calculation of the "Percent Identity" and the "Similarity" between the two sequences. The numbers calculated using UWGCG package version 9.1 is slightly different from the version 8.
The term "position" is in the context of the present invention to be understood as the number of an amino acid in a peptide or polypeptide when counting from the N- terminal end of said peptide/polypeptide. The position numbers used in the present invention refer to different subtilases depending on which subgroup the subtilase belongs to. As m entioned above the alkaline subtilases KP43, KP1790, KP9860, Y, SD-521 and E1 belong to the JP170 subgroup, based on sequence homology. Due to the exten- sive homology only subtilase Ya and SD-521 are in Fig. 1 aligned with JP170. The JP170 subtilase, Y subtilase and SD-521 subtilase are numbered according to SEQ ID NO:1 , SEQ ID NO:2 and SEQ ID NO:3, respectively.
The invention, however, is not limited to variants of these particular subtilases but extends to parent subtilases, especially of the JP170 type, containing amino acid residues at positions which are "equivalent" to the particular identified residues in the JP170 subtilase.
A residue (amino acid) position of a JP170 type subtilase is equivalent to a residue (position) of the JP170 subtilase, if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in the JP170 subtilase (i.e., having the same or similar functional capacity to combine, react, or interact chemically).
In order to establish homology to primary structure, the amino acid sequence of a precursor protease is directly compared to the JP170 subtilase primary sequence by aligning t he a mino a cid s equence of an i solated o r p arent wild type e nzyme with a suitable well-known (standard) enzyme of the same group or class of enzymes to define a frame of reference. This type of numbering has been used in numerous patent applications relating to subtilisins of the I-S1 and I-S2 subgroups with subtilisin BPN' as the standard subtilisin. If nothing else is indicated herein, in the present instance the JP170 subtilase has been chosen as standard.
In order to establish homology to the tertiary structure (3D structure) of JP170, the 3D structure based a lignment i n F ig. 1 h as been provided. By u sing this a lignment the amino acid sequence of a precursor JP170 type subtilase may be directly correlated to the JP170 primary sequence. For a novel JP170 type subtilase, the (3D based) position corresponding to a position in JP170 is found by i) identifying the JP170 type subtilase from the alignment of Fig. 1 that is most homologous to the novel sequence, ii) aligning the novel sequence with the sequence identified to find the correspond- ing position in the JP170 type subtilase from Fig. 1 , and iii) establishing from Fig. 1 the corresponding position in JP170.
For comparison and finding the most homologous sequence the GAP program from GCG package as described below are used. The alignment can as indicated above be obtained by the GAP routine of tl — ie GCG package version 8 to number the variants u sing the following parameters: g ap c__. reation penalty = 3 and gap extension penalty = 0.1 and all other parameters kept at their default values. The alignment may define a number of deletions and insertions in relation to the sequence of JP170. In the alignment deletions are indicated by asterixes (*) in the referenced sequence, and the referenced enzyme will be considered to have a gap at thie position in question. Insertions are indicated by asterixes (*) in the JP170 sequence, and the positions in the referenced enzyme are given as the position number of the last amino acid residue where a corresponding amino acid residue exists in the standard enzyme with a lower case letter appended in alphabetical order, e.g. 82a, 82b, 82c, 82d.
In case the referenced enzyme contains a N- or C-terminal extension in comparison to JP170; an N-terminal extension is given the position number 0a, 0b, etc. in the di rection of the N-terminal; and a C-terminal extension will be given either the position number of the C-terminal amino acid residue of JP170 with a lower case letter appended in alphabetical order, or simply a continued consecutive numbering.
Thus for comparisons JP170 type subtilases are numbered by reference to the positions of the JP170 subtilase (SEQ ID NO: 1 ) as provided in Fig. 1. The position is then indicated as "corresponding to JP170". Subtilases belonging to the BPN' subgroup refers to the positions of Subtilisin Novo
(BPN') from β. amyloliquefaciens (SEQ ID NO:4).
Subtilases belonging to the TY145 subgroup refers to the positions of the TY145 subtilase (SEQ ID NO:7), see also PCT/DK2004/000066.
DETAILED DESCRIPTION OF THE INVENTION
Despite the great homology of the subtilases described above the inventors of the present invention have elucidated the three-dimensional structure of JP170, SEQ ID NO:1 by X-ray crystallography and found that there are several differences between this and the three-dimensional structure of BPN'. The inventors of the present invention have further compared the sequence homology of subtilases belonging to the Subtilisin subgroup. This is shown in Figure 3 of the present invention.
On the basis of this comparison the inventors of the present invention suggest to divide the Subtilisin subgroup so that the JP170 type subtilases become a separate subgroup in addition to the subgroups of BPN' subtilases and TY145 subtilases PCT/DK2004/000066. JP170 type subtilases
The term "JP170 subtilase" or "JP170 type subtilase" should in the context of the present invention be understood as a subtilase belonging to the Subtilisin group according to Siezen et al. Protein Science 6 (1997) 501-523 and which has at least 58% homology to JP170, SEQ ID NO:1. In particular a JP170 type subtilase may have at least 60% homology to SEQ ID NO:1 , such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to JP17O, i.e. to SEQ ID NO:1. Thus, among others the alkaline proteases KP43, KP1790, KP9860, Prote- ase Ya, Protease E-1 and SD-521 are subtilases belonging to the JP170 subgroup of subtilases.
A JP170 subtilase suitable for the purpose described herein m ay be a subtilase homologous to the three-dimensional structure of JP170, i.e. it may be homologous to the three-dimensional structure defined by the structure coordinates in Appendix 1. As it is well-known to a person skilled in the art that a set of structure coordinates for a protein or a portion thereof is a relative set of points that define a shape in three dimensions, it is possible that an entirely different set of coordinates could define an identical or a similar shape. Moreover, slight variations in the individual coordinates may have little or no effect on the overall shape. These variations in coordinates may be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates of JP170 (Appendix 1 ) may be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above. Alternatively, said variations may be due to differences in the primary amino acid sequence.
If such variations are within an acceptable standard error, such as 0.8 A, as compared to the structure coordinates of Appendix 1 , said three-dimensional structure is within the context of the present invention to be understood as being homologous to the structure of Appendix 1. The standard error may typically be measured as the root mean square deviation of e.g. conserved backbone residues, where the term "root mean square deviation" (RMS) means the square root of the arithmetic mean of the squares of the deviations from the mean.
As it is also well-known to a person skilled in the art that within a group of proteins which have a homologous structure there may be variations in the three-dimensional structure in certain areas, sub-structures or domains of the structure, e.g. loops, which are not or at least only of a small importance to the functional domains of the structure, but which may result in a big root mean square deviation of the conserved residue backbone atoms between said structures.
Thus it is well known that a set of structure coordinates is unique to the crystallised protein. No other three dimensional structure will have the exact same set of coordinates, be it a homologous structure or even the same protein crystallised in a different manner. There are natural fluctuations in the coordinates. The overall structure and the inter-atomic relationship can be found to be similar. The similarity can be discussed in terms of root mean square deviation of each atom of a structure from each "homologous" atom of an- other structure. However, only identical proteins have the exact same number of atoms. Therefore, proteins having a similarity below 100% will normally have a different number of atoms, and thus the root mean square deviation can not be calculated on all atoms, but only the ones that are considered "homologous". A precise description of the similarity based on the coordinates is thus difficult to describe and difficult to compute for homolo- gous proteins. Regarding the present invention, similarities in 3D structure of different subtilases can be described by the content of homologous structural elements, and/or the similarity in amino acid or DNA sequence. For sequences having no deletions or insertions a RMS for the CA carbon atoms can be calculated. Optionally a JP170 type subtilase is further characterised as comprising the following structural characteristics: a) a twisted beta-sheet with 7 strands, b) six alpha helices, c) at least three ion-binding sites, and not comprising the Strong and Weak ion-binding site of the BPN' like subtilases Further the isolated nucleic acid sequence encoding a JP170 subtilase of the invention hybridizes with a complementary strand of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:1 preferably under low stringency conditions, at least under medium stringency conditions, at least under medium/high stringency conditions, at least under high stringency conditions, at least under very high stringency conditions. Suitable experimental conditions for determining hybridization at low, medium, or high stringency conditions between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the filter in a solution of 5 x SSC, 5 x Denhardt's solution (Sambrook et al. 1989), 0.5 % SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a concentra- tion of 10ng/ml of a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13), 32P-dCTP-labeled (specific activity > 1 x 109 cpm/μg ) probe for 1 2 hours at ca. 45°C. The filter is then washed twice for 30 minutes in 2 x SSC, 0.5 % SDS at least * 55°C (low stringency), more preferably at least 60°C (medium stringency), still more pref- erably at least 65°C (medium/high stringency), even more preferably at least 70° C (high stringency), and even more preferably at least 75°C (very high stringency).
BPN' subtilases
A BPN' subtilase or BPN' type subtilase is in the context of the present invention to be understood as a subtilase belonging to the Subtilisin group according Siezen et al. Siezen et al. Protein Science 6 (1997) 501-523 and which has at least 61% homology to SEQ ID NO:4. In particular a BPN' subtilase may have at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to BPN', i.e. to SEQ ID NO:4.
Further the isolated nucleic acid sequence encoding a BPN' subtilase of the invention hybridizes with a complementary strand of the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:4 preferably under low stringency conditions, at least under medium stringency conditions, at least under medium/high stringency conditions, at least under high stringency conditions, at least under very high stringency conditions.
In one embodiment of the present invention a BPN' subtilase suitable for the purpose described herein may be a subtilase homologous to the three-dimensional structure of BPN' as defined by the structure coordinates given in PDB Nos. 1SBT and 1GNS (Protein Data Bank), or one of the several other structures of BPN' that are accessible from the Protein Data Bank. Variations between homologous structures may occur for several reasons as described above. Thus a BPN' subtilase within the context of the present invention is to be understood as any subtilase having the structural characteristics pertaining to the BPN' subtilases as described above, and in addition such subtilases do preferably not have further structural characteristics which are not present in the BPN' subtilases as de- scribed herein. In the context of the present invention a BPN' type subtilase has two ion- binding sites. A BPN' like subtilase may, in the context of the present invention, belong to branch l-S of the subtilisins i.e. to branch I-S1 , the "true" subtilisins or I-S2, the highly alkaline proteases (Siezen et al., Protein Engng. 4 (1991 ) 719-737).
Examples of BPN' type subtilases include the subtilisin 309 (PDB NO:1SVN, SAVI- NASE®, NOVOZYMES A/S) and subtilisin Carisberg (ALCALASE®, NOVOZYMES A/S), among others. In connection with Figure 1 of R.J. Siezen and J.A.M Leunissen (Protein science, Vol. 6 (3), pp. 501-523, 1997) page 502 a structure of subtilases is described as: A subtilase consists of 6-8 helices, 11 strands of which 7 are central in a twisted beta-sheet. Two ion-binding sites are mentioned, the so called "Strong" and "Weak" calcium-binding sites. It was later discovered that for some structures (subtilisin DY PDB no. 1BH6, 1998), the Weak calcium-binding site was shown to be a Na (sodium) binding site when the calcium concentration in the crystallization medium was low. Thus, in the following we refer to ion- binding sites instead of calcium-binding sites.
TY145 subtilases
A TY145 subtilase or TY145 type subtilase is in the context of the present invention to be understood as a subtilase which has at least 63% homology to SEQ ID NO:7. In particular said TY145 subtilase may have at least 65%, such as at least 70%, at least 74%, at least 80%, at least 83%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to TY145, i.e. to SEQ ID NO:7.
In one embodiment of the present invention a TY145 subtilase suitable for the purpose described herein may be a subtilase homologous to the three-dimensional structure of TY145 as defined by the structure coordinates given in PCT/DK2004/000066. Variations between homologous structures may occur for several reasons as described above. Thus a TY145 subtilase within the context of the present invention is to be understood as any subtilase having the structural characteristics pertaining to the TY145 subtilases as described above, and in addition such subtilases do preferably not have further structural characteristics which are not present in the TY145 subtilases as described herein. Typically a TY145 subtilase further comprises the following structural characteristics: a) a twisted beta-sheet with 7 strands, b) six alpha helices, c) at least three ion-binding sites, wherein the Strong ion-binding site of the BPN' type sub- tilases is not present,
Examples of s ubtilases of the TY145 type i nclude the TY145 s ubtilase, the psychrophilic subtilisin protease S41 derived from the Antarctic Bacillus TA41 , herein also called TA41 subtilase (Davail S et al., 1994, J. Biol. Chem., 269, 17448-17453), and the psychrophilic subtilisin protease S39 derived from the Antarctic Bacillus TA39, herein also called TA39 subtilase (Narinx E et al., 1997, Protein Engineering, 10 (11 ), 1271-1279). Three-dimensional structure of JP170 subtilases
The JP170 subtilase was used to elucidate the three-dimensional structure forming the basis for the present invention.
The structure of JP170 was solved in accordance with the principle for x-ray crystallographic methods, for example, as given in X-Ray Structure Determination , Stout, G.K. and Jensen, L.H., John Wiley & Sons, Inc. NY, 1989.
The structural coordinates for the solved crystal structure of JP170 are given in standard PDB format (Protein Data Bank, Brookhaven National Laboratory, Brookhaven, CT) as set forth in Appendix 1. It is to be understood that Appendix 1 forms part of the pre- sent application. In the context of Appendix 1 , the following abbreviations are used: CA refers to c-alpha (carbon atoms) or to calcium ions, (however to avoid misunderstandings we normally use the full names "c-alpha atoms" and "calcium" or "ion" in the present specification). Amino acid residues are given in their standard three-letter code. The attached structural coordinates contain the protease structure, and an inhibitor structure CI2 as well as water molecules. The protease coordinates has a chain identification called A, whereas the CI2 inhibitor is called B, the calcium ions are called C, and the water is W. In the following the positions of the mentioned residues refer to the sequence of JP170 as disclosed in SEQ ID NO:1.
The JP170 structure consists of two domains, a catalytic domain and a C-terminal domain.
The structure of the catalytic domain shows the same overall fold as found in the S8 family of subtilisins. The structure comprises a twisted beta-sheet with 7 strands arranged in the following sequential order S2, S3, S1 , S4, S5, S6, S7.
There are six alpha helices in the catalytic domain structure of which number H1 contains residues 9-17, H 2 contains residues 68-76, H3 contains residues 1 10-1 19, H 4 contains residues 139-150, H5 contains residues 253-273 and H6 contains residues 281- 291.
The C-terminal domain comprises a strand motif, a so called "beta sandwich" consisting of sheets a and b. The sheet in this domain is combined of strands in an anti- parallel fashion, whereas the strand in the catalytic domain is combined in parallel. The sequential order of the strands can be denoted as: S1a-S1b-S3a-S3b-S4b-S4a-S2b-S2a with the beta sandwich organised as to the two sheets S1a, S3a, S4a, S2a and S1 b, S3b, S4b, S2b.
The JP170 subtilases were found to lack the well-known Strong and Weak ion- binding sites of the BPN' subtilases. However, the JP170 subtilases have three ion-binding sites which are not present in the BPN' subtilisin structures. This can be seen in the struc- tural alignment presented in Fig. 2. These three ion-binding sites are hereinafter referred to as Site 1 , which is placed in the catalytic domain, and Site 2 and 3 which are placed in the non-catalytic C-terminal domain.
Thus in relation to the atomic coordinates disclosed in Appendix 1 , the ion— binding sites of JP170 are located at:
Site 1 - calcium atom named A601 CA
Site 2 - calcium atom named A603 CA, and
Site 3 - calcium atom named A602 CA in the PDB table (Appendix 1).
The position of an ion-binding site can be defined by the distance to four specific atoms in the core structure. The distance from the ion-binding site to the c-alpha atoms of the three active site residues has been chosen. Throughout the subtilases the residues Ser, His and Asp in the active site are highly conserved. In JP170 they are Asp30, His68 and Ser254. The fourth distance chosen is the distance to the c-alpha atom of the arnino acid residue coming first after the active site serine residue in the sequence (herein after called "next to Ser"); in the 3D structure of JP170 it is Met255. o
In a preferred embodiment of the present invention, the distance between: a) ion-binding site 1 and i) Asp c-alpha atom is 26.70-28.7θA, ii) His c-alpha atom is 22.10-24.10A, iii) Ser c-alpha atom is 16.95-18.95A, iv) next to Ser c-alpha atom is 15.30-17.30A, b) ion-binding site 2 and i) Asp c-alpha atom is 33.50-35.5θA, ii) His c-alpha atom is 37-39A, iii) Ser c-alpha atom is 29.40-31.40A, iv) next to Ser c-alpha atom is 30.70-32.70A, c) ion-binding site 3 and i) Asp c-alpha atom is 41.50-43.50A, ii) His c-alpha atom is 42.90-44.90A, iii) Ser c-alpha atom is 34.50-36.50A, iv) next to Ser c-alpha atom is 35-37A.
Below the specific distances between the four chosen c-alpha atoms and the three ion binding sites of the JP170 subtilase; and the distances between the ion binding sites are given in A: site 1 site 2 site 3
Asp30 27.69 34.49 42.48
His68 23.12 38.03 43.87
Ser254 17.95 30.41 35.51
Met255 16.34 31.68 36.02 site 1 0 35.29 32.92 site 2 35.29 0 14.08 site 3 32.92 14.08 0
However, these distances may vary from one subtilase to the other. The present distances are given with a calcium ion in the structure. If a sodium ion was bound instead the distances would be shifted a little bit. Generally the distances can vary ±0.8θA, pref- erably ±0.7θA, ±0.6θA, ±0.5θA, ±0.4θA, or most preferably ±0.3θA.
Further, in the JP170 like subtilases, the peptide structure circumscribing ion- binding site 1 up to a distance of 10 A from the metal ion is composed of the amino acid residues placed in positions 183-189, 191-204 and 224-225.
The peptide structure circumscribing ion-binding site 2 up to a distance of 10 A from the metal ion is composed of residues 378-393.
The peptide structure circumscribing ion-binding site 3 up to a distance of 10 A from the metal ion is composed of residues 348, 350, 352, 363-370, 380-383, 391-400 and 414- 420.
Comparison to the I-S1 and I-S2 subgroups (BPN' like subtilases)
In comparison to the BPN' like subtilase structures the structure of the JP170 like subtilases can be divided into a "core subtilase-like" region, an "intermediate" region and a "nonhomologous" region.
The active site can be found in the core subtilase-like region, which is structurally closely related to the BPN' structures. The core subtilase-like region is composed of residues 17-34, 197-209 and 216-232, and contains the alpha-helix H3 and the central alpha- helix H5 in which the active site serine residue is situated in the N-terminal part. The core subtilase-like region has an RMS lower than 1.2.
Outside the core subtilase-like region the structure of the JP170 like subtilase dif- fers from the BPN' structures to a greater extent.
The intermediate region consists of residues 42-46, 150-186, 245-272 and 278- 296. The intermediate region has an RMS bigger than 1.2 and less than 1.8. The relationships between the three-dimensional structure and functionality are potentially difficult to predict in this region of the JP170 like subtilases.
The nonhomologous region consists of residues 1-16, 35-41 , 47-149, 187-196, 210-215, 233-244, 273-277 and 297-316. The nonhomologous region has a RMS h igher than 1.8. The relationships between the three-dimensional structure and functionality are very difficult to predict in this region of the JP170 like subtilases.
Many loops in the 3D structure of the JP170 like subtilases differ significantly from the BPN' type structures, both in length and in content of amino acid residues. The following loops or protein sequence stretches of JP170 are compared to Savinase (BPN' numbering in parenthesis, (cf. Fig. 4)): G32-H43 (G34-H39) E44-Y54 (P40-A48) G57-G67 (V51-G63) N79-N82 (I75-V81 ) I96-P107 (V95-S105) A108-S119 (106-N117) A131-Y137 (S128-S132) T138-D152 (A133-G146) E162-M69 (S156-M 65) G173-T180 (A169-A176) E185-N199 (D181-N184) G208-D218 (G193-D197) S232-K246 (G21 1-T213) D294-N303 (S256-L262)
The loops N79-N82 (I75-V81 ) and G208-D218 (G193-D197) are in contact with a ion-binding site in Savinase, but not in JP170. Similarly the loop E185-N199 (D181-N 184) is in contact with a ion-binding site in JP170, but not in Savinase. This knowledge opens for possibilities of adding or removing ion-binding sites to subtilases of the JP170 and BPN' like types. A good example of the difference is the loop S232-K246 in JP170 which has 15 residues compared to the corresponding BPN' type loop G211-T213 (in Savinase), which has only three residues. In the JP170 like subtilases, the loop folds back to the substrate binding site, especially the P' parts of the substrate binding site. The loop is situated close to the substrate as illustrated by the CI2 inhibitor bound in the 3D structure attached (Ap- pendix l ).
The location of loop S232-K246 in JP170 can be described in relation to the four specific residues as described above. The distance from the CA atom of residue W240 in the loop to the CA atoms of the active site residues are: Residue H68 D30 S254 M255
Distance, A 11.45 18.51 13.06 11.94
As mentioned above, distances like these can vary ±0.8θA, preferably ±0.7θA, ±0.6θA, ±0.5θA, ±0.4θA, or most preferably ±0.3θA.
Furthermore, distances from the residues of JP170 loop S232-K246 to atoms of the CI2 inhibitor can be calculated. These distances are: from CA atom of W240 to CA atom of R62 in CI2 is 7.49A, from CA atom of F239 to CA atom of R62 in CI2 is 8.39A, from CA atom of S238 to CA atom of R62 in CI2 is 8.42A, from CA atom of S237 to CA atom of R62 in CI2 is 9.44A, from CA atom of S238 to CA atom of E60 in CI2 is 9.42A.
The distances from JP170 active site residue S254 to atoms of the CI2 inhibitor, as placed in the 3D coordinates of Appendix 1 , are: from CA atom of S254 to CA atom of E60 in CI2 is 5.25A, from CA atom of S254 to CA atom of R62 in CI2 is 11.55A, from CA atom of S254 to CA atom of T58 in CI2 is 7.06A, from CA atom of S254 to CA atom of M59 in CI2 is 4.71 A. The distances can vary ±0.8θA, preferably ±0.7θA, ±0.6θA, ±0.5θA, ±0.4θA, or most preferably ±0.3θA.
A preferred JP170 like subtilase variant has a deletion in the region S232-K246, and the subsequent insertion of one or more residues to partly or completely remove the loop. Preferred variants comprises the deletion of L233-S245 + insertion of Asn, deletion of L233-D244 + insertion of Gly or deletion of S232-D244 + insertion of Gly. Similar considerations can be made in respect of differences to the TY145 structure.
Homology building of JP170, BPN' and TY145 subtilases A model structure of a JP170 type subtilase, a BPN' type subtilase or a TY145 type subtilase can be built using the Homology program or a comparable program, e.g., Modeller (both from Molecular Simulations, Inc., San Diego, CA). The principle is to align the amino acid sequence of a protein for which the 3D structure is known with the amino acid sequence of a protein for which a model 3D structure has to be constructed. The structur- ally conserved regions can then be built on the basis of consensus sequences. In areas lacking homology, loop structures can be inserted, or sequences can be deleted with subsequent bonding of the n ecessary residues using, e .g., the program Homology. S ubsequent relaxing and optimization of the structure should be done using either Homology or another molecular simulation program, e.g., CHARMm from Molecular Simulations.
Methods for designing JP170, BPN', and TY145 subtilase variants
Comparisons of the molecular dynamics of different proteins can give a hint as to which domains are important or connected to certain properties pertained by each protein. Thus the present invention relates to a method for constructing a variant of a parent subtilase, wherein the variant has at least one altered property as compared to said parent subtilase, which method comprises: a) analyzing the three-dimensional structure of the parent subtilase to identify, on the basis of an evaluation of structural considerations in relation to a JP170 three di- mensional structure, at least one amino acid residue or at least one structural region of the subtilase, which is of relevance for altering said property; b) modifying the DNA of the polynucleotide encoding the parent to construct a polynucleotide encoding a variant subtilase, which in comparison to the parent subtilase, has been modified by deletion, substitution or insertion of the amino acid residue or structural part identified in i) so as to alter said property; c) expressing the variant subtilase in a suitable host, and d) testing the resulting subtilase variant for said property.
More specifically the invention relates to a method of producing a subtilase variant, wherein the variant has at least one altered property as compared to a parent subtilase, which method comprises: a) producing a model structure of the parent subtilase on the three-dimensional structure of BPN', TY145 or JP170; or producing an actually determined three- dimensional structure of the parent subtilase, b) comparing the model or actual three-dimensional structure of the parent subtilase to the JP170 structure by superimposing the structures through matching the CA, CB, C, O, and N atoms of the active site residues, c) identifying on the basis of the comparison in step b) at least one structural part of the parent subtilase, wherein an alteration in said structural part is predicted to re- suit in an altered property; d) modifying the n ucleic a cid sequence e ncoding the parent subtilase to produce a nucleic acid sequence encoding at least one deletion or substitution of one or more amino acids at a position corresponding to said structural part, or at least on e insertion of one or more amino acid residues in positions corresponding to said struc- tural part; e) performing steps c) and d) iteratively N times, where N is an integer with the value of one or more; f) preparing the variant resulting from steps a) - e); g) testing the properties of said variant; and h) optionally repeating steps a) - g) recursively; and i) selecting a subtilase variant having at least one altered property as compared to the parent subtilase. j) expressing the modified nucleic acid sequence in a host cell to produce the variant subtilase; k) isolating the produced subtilase variant;
I) purifying the isolated subtilase variant and m) recovering the purified subtilase variant.
The present invention thus generally relates to the use of the JP170 structure as provided herein for the identification of desired modifications in subtilases of any of the three subtilisin types, the BPN' types (I-S1 and I-S2 subgroups), the TY145 types and the JP170 types through modelling the 3-D structure of a parent subtilase to the type it belongs to and subsequent comparison thereof to the JP170 3-D structure, or in instances where the 3-D structure of the parent subtilase is actually known by comparison thereof to the JP170 3-D structure.
Based on this comparison at least one residue in the parent subtilase is selected for modification by substitution, deletion or insertion in order to provide a subtilase variant with altered properties in comparison to the parent subtilase.
In one embodiment the parent subtilase may therefore belong to the sub-group I- S1 , preferably selected from the group consisting of ABSS168, BASBPN, BSSDY, and BLSCAR, or functional variants thereof having retained the characteristic of sub-group I- S1.
In another embodiment the parent subtilase belongs to the sub-group I-S2, preferably selected from the g roup consisting of BLS147, BLS309, BAPB92, and BYSYAB, or functional variants thereof having retained the characteristic of sub-group I-S2.
In a further embodiment the parent subtilase belongs to the TY145 type subgroup, preferably selected from the group comprising TY145, protease S41 also called TA41 protease S39 also called TA39 subtilase, etc.
Specifically the parent subtilase belongs to the JP170 type subgroup, preferably selected from the group comprising JP170, KP43, KP9860, Protease E-1 , Protease Ya, Protease SD-521 , etc.
A further embodiment of the invention relates to a method of producing a JP170 type subtilase variant, wherein the variant has at least one altered property as compared to a parent subtilase, which method comprises: a) producing a model structure of the parent JP170 type subtilase on the three- dimensional structure of JP170; or producing an actually determined three- dimensional structure of the parent subtilase, b) comparing the model or actual three-dimensional structure of the parent JP170 type subtilase to the BPN' or TY145 structure by superimposing the structures through matching the CA, CB, C, O, and N atoms of the active site residues, c) identifying on the basis of the comparison in step b) at least one structural part of the parent JP170 type subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent JP170 type subtilase to produce a nucleic acid sequence encoding at least one deletion or substitution of one or more amino acids at a position corresponding to said structural part, or at least one insertion of one or more amino acid residues in positions corresponding to said structural part; e) performing steps c) and d) iteratively N times, where N is an integer with the value of one or more; f) preparing the JP170 type subtilase variant resulting from steps a) - e); g) testing the properties of said variant; and h) optionally repeating steps a) - g) recursively; and i) selecting a JP170 type subtilase variant having at least one altered property as compared to the parent subtilase. j) expressing the modified nucleic acid sequence in a host cell to produce the variant subtilase; k) isolating the produced JP170 type subtilase variant;
I) purifying the isolated subtilase variant and m) recovering the purified subtilase variant.
The invention also comprises the protease variants produced by the above meth- ods.
Stability - alteration of ion-binding sites
As described above the three-dimensional structure of JP170 subtilases as provided in Appendix 1 indicates the presence of three ion-binding sites not present in the BPN' subtilisin structures, thus lacking the Strong and Weak ion-binding site of the BPN' subtilases. Stability of ion-binding sites is important for the functionality of the enzyme. Therefore alterations of the ion-binding sites are likely to result in alterations of the stability of the enzyme.
Improved stability
Stabilisation of a JP170 s ubtilase may possibly be o btained by a lterations i n the positions close to the ion-binding sites. Thus in one embodiment of the method of the invention step (c) above identifies amino acid residue positions located at a distance of 1θA or less to the ion-binding site of the JP170 type parent, preferably positions located at a distance of 6 A or less.
Thus a preferred variant of the present invention has a modification in one or more of the positions located at a distance of 1θA to the ion-binding sites of JP170 (SEQ ID NO:1). These positions are:
Site 1 : 183-189 (i.e. positions 183, 184, 185, 186, 187, 188, 189),
191-204 (i.e. positions 191 , 192, 193, 194, 195, 196, 197, 198, 199,
200, 201 , 202, 203, 204), 224-225;
Site 2: 378-393 (i.e. positions 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387,
388, 389, 390, 391 , 392, 393);
Site 3: 348, 350, 352, 363-370 (i.e. positions 363, 364, 365, 366, 367, 368, 369, 370),
380-383 (i.e. positions 380, 381 , 382, 383),
391-400 (i.e. positions 391 , 392, 393, 394, 395, 396, 397, 398, 399, 400),
414-420 (i.e. positions 414, 415, 416, 417, 418, 419, 420).
Corresponding positions in other JP170 type subtilases may be identified as disclosed above or by using Fig. 1 herein.. In detergent compositions calcium chelaters contribute to removal of calcium from the subtilases with subsequent inactivation of the enzyme as the result. To decrease the inactivation due to calcium removal of e.g. calcium chelaters variants with improved calcium stability was constructed. Preferred variants stabilised in ion-binding site 1 are S193Q.Y; H200D.N and
H200D.N+D196N.
Preferred variants stabilised in ion-binding site 2 are N390D and N391 D, and preferred variants stabilised in ion-binding site 3 are G394N,Q,F,Y,S and W392S,N,Q.
Alteration of thermostability
A variant with improved stability (typically increased thermostability) may be obtained by substitution with proline, introduction of a disulfide bond, altering a hydrogen bond contact, altering charge distribution, introduction of a salt bridge, filling in an internal structural cavity with o ne o r m ore a mino acids with b ulkier s ide groups ( in e.g. regions which are structurally mobile), substitution of histidine residues with other amino acids, removal of a deamidation site, or by helix capping.
Regions with increased mobility:
The following regions of JP170 have an increased mobility in the crystal structure of the enzyme, and it is presently believed that these regions can be responsible for stability or activity of JP170. Especially thermostabilisation may possibly be obtained by altering the highly mobile regions. Improvements of the enzyme can be obtained by mutation in the below regions and positions. Introducing e.g. larger residues or residues having more atoms in the side chain could increase the stability, or, e.g., introduction of residues having fewer atoms in the side chain could be important for the mobility and thus the activity profile of the enzyme.
Two methods are used extract the highly mobile regions from a 3D structure. One is a molecular dynamics calculation of the isotropic fluctuations by using the program CHARMm from MSI (Molecular Simulations Inc.), and the other is an analysis of the B-factors. The B- factors are listed in Appendix 1 and give a value to the uncertainty of determination of the location of the various atoms of the structure. The uncertainty relates to the mobility of the atoms in the molecules in the crystal lattice. This mobility reflects the thermal motion of the atoms and thus indicates possible sites for thermostabilisation of the enzyme.
Thus, by analysing the B-factors taken from the coordinate file in Appendix 1 , (see "X-Ray Structure D etermination, Stout, G.K. and Jensen, L.H., John W iley & Sons, Inc. NY, 1989") the following mobile regions in the JP170 structure were determined: 13-18 (i.e. positions 13, 14, 15, 16, 17, 18),
37-43 (i.e. positions 37, 38, 39, 40, 41 , 42, 43),
47-50 (i.e. positions 47, 48, 49, 50),
57-59 (i.e. positions 57, 58, 59),
96-103 (i.e. positions 96, 97, 98, 99, 100, 101 , 102, 103),
131-134 (i.e. positions 131 , 132, 133, 134),
152-153
162-166 (i.e. positions 162, 163, 164, 165, 166),
188-195 (i.e. positions 188, 189, 190, 191, 192, 193, 194, 195),
210
234-246 (i.e. positions 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245,
246),
372-378 (i.e. positions 372, 373, 374, 375, 376, 377, 378),
387-392 (i.e. positions 387, 388, 389, 390, 391 , 392),
406-407
419.
Molecular dynamics simulations at 300K and 400K of JP170 provided the following highly mobile regions:
37-42 (i.e. positions 37, 38, 39, 40, 41 , 42),
57-60 (i.e. positions 57, 58, 59, 60),
66-67,
98-103 (i.e. positions 98, 99, 100, 101 , 102, 103),
107-111 (i.e. positions 107, 108, 109, 110, 111),
188-193 (i.e. positions 188, 189, 190, 191 , 192, 193),
236-240 (i.e. positions 236, 237, 238, 239, 240),
326-332 (i.e. positions 326, 327, 328, 329, 330, 331 , 332),
337-342 (i.e. positions 337, 338, 339, 340, 341 , 342),
355-360 (i.e. positions 355, 356, 357, 358, 359, 360),
372-377 (i.e. positions 372, 373, 374, 375, 376, 377),
384-388 (i.e. positions 384, 385, 386, 387, 388),
404-411 (i.e. positions 404, 405, 406, 407, 408, 409, 410, 411).
Thus, a preferred JP170 subtilase variant of the present invention has been modified in one or more of the above mentioned positions of SEQ ID NO:1. Further preferred variants comprises one or more alterations in the regions 57-60, 66-67, 107-1 1 1 , 236-240, 326-332, 355-360, 372-377, 384-388, 404-41 1. Especially preferred is variant W240H.Y and variants modified in the region 355-360, such as variants comprising one or more of the modifications: G355A.S; S356T.N; T357N,Q,D,E,P; T358S; A359S,T,N,Q and S360T.N.
Variants modified in the region 355-360 may be produced in accordance with the method for random mutagenesis by use of the DOPE program as described herein. To obtain variants comprising 1-3 modifications in region 355-360 one may introduce the substitutions with the following frequencies: wild-type modified
95% 5% G355A.S
90% 10% S356T.N
80% 20% T357N,Q,D,E,P
90% 10% T358S
80% 20% A35S9S,T,N,Q
80% 20% S360T.N.
Disulfide bonds:
A JP170 variant of the present invention with improved stability, e.g. thermostability, as compared to the parent JP170 subtilase may be obtained by introducing new inter- domain or intra-domain bonds, such as by establishing inter- or intra-domain disulfide bridges.
Thus a further aspect of the present invention relates to a method for producing a variant of a parent JP170, wherein step (c) identifies amino acid residue positions in the parent JP170 type subtilase, the modification of which may create at least one disulfide bridge by insertion of or substitution with at least one Cys residue.
The below mentioned amino acid residues identified in the amino acid sequence of SEQ ID NO:1 are considered as being suitable for cysteine replacement. With one or more of these substitutions with cysteine, disulfide bridges may possibly form in a variant of JP170. The substitutions are: G21C+A86C, V26C+A265C, G57C+G105C, G74C+A229C, Q111C+N143C, G160C+S170C, A286C+V349C, A27C+A122C, A45C+G78C, V72C+P258C, G78C+A229C, D98C+G104C, Q111C+Y147C, G135C+G167C, R142C+P354C, V144C+A178C, G182C+P217C, A183C+G223C, A195C+Y225C, F271C+P279C, A287C+A430C, A293C+T310C, E322C+S428C, S324C+A332C, S327C+P424C, D352C+N397C, G355C+T362C, G291C+S314C. Preferred variants comprise one or more of the substitutions: G21C+A86C,
V26C+A265C, G57C+G105C, G74C+A229C, Q111C+Y143C, G160C+S170C, A286C+V349C, A4C+P222C and A27C+A117C.
Similar residues s uitable for cysteine replacement i n s ubtilases h omologσus with JP170 can be elucidated by finding the homologous positions in the alignment of Figure 1. Concerning another JP170 like sequence the homologous positions suitable for cysteine replacement can be selected by aligning said JP170 like sequence with all of the sequences of Figure 1 using the GAP analysis m ethod as described above. The suitable residues can then be selected in accordance with the homologous positions in the most homologous of SEQ ID's NO:1 , 2 and 3 which are the sequences of the subtilases aligned in Figure 1.
Surface charge distribution
A variant with improved stability (typically improved thermostability) as compared to the parent subtilase may be obtained by changing the surface charge distribution of the subtilase. For example, when the pH is lowered to about 5 or below histidine residues typically become positively charged and, consequently, unfavorable electrostatic interactions on the protein surface may occur. By engineering the surface charge of the subtilase one may avoid such unfavorable electrostatic interactions that in turn lead to a higher stability of the subtilase.
Charged amino acid residues are (a) positively charged: Lys, Arg, His (pH<5), Tyr (pH>9) and Cys (pH>10) and (b) negatively charged: Asp and Glu.
Therefore, a further aspect of the present invention relates to method for constructing a variant of a parent subtilase, the method comprising: a) identifying, on the surface of the parent subtilase, preferably a JP170 like or a BPN' like subtilase, at least one amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His; b) substituting, on the surface of the parent subtilase, at least one amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His with an uncharged amino acid residue; c) optionally repeating steps a) and b) recursively; d) optionally, making alterations each of which is an insertion, a deletion or a substitution of an amino acid residue at one or more positions other than b); e) preparing the variant resulting from steps a) - d); f) testing the stability of said variant; and g) optionally repeating steps a) - f) recursively; and h) selecting a subtilase variant having increased stability as compared to the parent subtilase. As will be understood by the skilled person it may also, in some cases, be advantageous to substitute an uncharged amino acid residue with an amino acid residue bearing a charge or, alternatively, it may in some cases be advantageous to substitute an amino acid residue bearing a charge with an amino acid residue bearing a charge of opposite sign. Thus, the above-mentioned method may easily be employed by the skilled person also for these purposes. In the case of substituting an uncharged amino acid residue with an amino acid residue bearing a charge the above-mentioned method may be employed the only difference being steps a) and b) which will then read: a) identifying, on the surface of the parent subtilase, at least one uncharged amino acid residue; b) substituting, on the surface of the parent subtilase, at least one uncharged amino acid residue with a charged amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His.
Also in the case of changing the sign of an amino acid residue present on the surface of the subtilase the above method may be employed. Again, compared to the above method, the only difference being steps a) and b) which, in this case, read: a) identifying, on the surface of the parent subtilase, at least one charged amino acid residue selected from the group consisting of Asp, Glu, Arg, Lys and His; b) substituting, on the surface of the parent subtilase, at least one charged amino acid residue identified in step a) with an amino acid residue having an opposite charge.
Thus, Asp may be substituted with Arg, Lys or His; Glu may be substituted with Arg,
Lys or His; Arg may be substituted with Asp or Glu; Lys may be substituted with Asp or Glu; and His may be substituted with Asp or Glu.
In order to determine the amino acid residues of a subtilase, which are present on the surface of the enzyme, the surface accessible area are measured using the DSSP pro- gram (Kabsch and Sander, Biopolymers (1983), 22, 2577-2637). All residues having a surface accessibilty higher than 0 0, 0.10, 0.20, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55 or 0.60 are regarded a surface residue.
Among the amino acid residues found on the surface of JP170 using the above method are N79, N316, L381 , K246, K9, K313 and K83. We consider the substitutions N79D, N316D and L381 D of particular interest for stabilisation by introduction of salt bridges, whereas the substitutions K246R, K9R, K313R and K83R are of particular interest for the stabilisation at high pH.
Similar substitutions may be introduced in equivalent positions of other JP170 like subtilases.
Substitution with proline residues
Improved thermostability of a subtilase can be obtained by subjecting the subtilase in question to analysis for secondary structure, identifying residues in the subtilase having dihedral angles φ (phi) and ψ (psi) confined to the intervals [-90°<φ<-40° and -180°<ψ<180°], preferably the intervals [-90°<φ<-40° and 120°<ψ<180°] or [-90°<φ<-40° and -50°<ψ<10°] and excluding residues located in regions in which the subtilase is characterized by possessing α- helical or β-sheet structure.
After the dihedral angles φ (phi) and ψ (psi) for the amino acids have been calculated, based on the atomic structure in the crystalline subtilases, it is possible to select position(s) which has/have dihedral phi and psi angles favorable for substitution with a proline residue. The aliphatic side chain of proline residues is bonded covalently to the nitrogen atom of the peptide g roup. The resulting cyclic five-membered ring consequently i mposes a rigid constraint on the rotation about the N-Cα bond of the peptide backbone and simultaneously prevents the formation of hydrogen bonding to the backbone N-atom. For these structural reasons, proline residues are generally not compatible with α-helical and β-sheet secondary con- formations.
If a proline residue is not already at the identified position(s), the naturally occurring amino acid residue is substituted with a proline residue, preferably by site directed mutagenesis applied on a gene encoding the subtilase in question.
In the group of JP170 type subtilases proline residues can advantageously be intro- duced at positions 22, 44, 110, 139, 140, 166, 198, 201 , 203, 231 , 282, 356, 357 and 378. Accordingly, a preferred JP170 variant has one or more of the substitutions: Q22P, E44P, L110P, T139P, D140P, S166P I198P, V201P, Q203P, S231P, S282P, S356P, T357P and K378P. Especially preferred are variants comprising one or more of: E44P, Q203P and S356P.
Improved activity of JP170 subtilases
As mentioned, the JP170 subtilases differ greatly from the BPN' like subtilases in having a long apparently non-catalytic C-terminal. A possible truncation of JP170 is the removal of approx. 115 residues including two ion-binding sites, which can be obtained by deletion of or within the region 311-433, which is the non-catalytic C-terminal. Preferred deletions com- prises the regions 317-433 or 315-433. Preferably the new C-terminal will be within the region of 311-325. Further, the deletion can be optimised with additional substitutions, such as one or more of L283N.Q; A290S.N and W306H,Y,K. Preferred truncations comprise: a) deletion of region 317-433 and the substitutions L283N + A290S + W306H, b) deletion of region 315-433 and the substitutions L283N + A290S + W306H.
Substrate binding site
The substrate binding site is identified by the residues in contact with a substrate model, such as the CI2 inhibitor. The 3D structure coordinates of the JP170 subtilase with CI2 bound in the active site are provided in Appendix 1. Without being limited to any theory, it is presently believed that binding between a substrate and an enzyme is supported by favorable interactions found within a sphere 10 A from the substrate molecule. Examples of such favorable bonds are hydrogen bonds, strong electrostatic interaction and/or hydrophobic interactions.
The following residues of the JP170 subtilase (SEQ ID NO:1 ), are within a distance of 1θA from the CI2 inhibitor which is bound to the substrate binding site. These residues are thus believed to be involved in interactions with said substrate: 29-32, (i.e. residues 29, 30, 31 , 32) 64-72, (i.e. residues 64, 65, 66, 67, 68, 69, 70, 71 , 72)
93,
96-98, (i.e. residues 96, 97, 98)
100-110, (i.e. residues 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110) 113-114, 127-136, (i.e. residues 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136) 138-141 , (i.e. residues 138, 139, 140, 141 ) 144, 157, 174,
180-183, (i.e. residues 180, 181 , 182, 183) 191 , 193-194, 202-207, (i.e. residues 202, 203, 204, 205, 206, 207) 211 ,
223-226, (i.e. residues 223, 224, 225, 226) 234-241 , (i.e. residues 234, 235, 236, 237, 238, 239, 240, 241 ) 249-258 (i.e. residues 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258). In an embodiment of the present invention a variant comprises a modification in one or more of the above mentioned positions. A preferred variant is W129L. JP170 with extra ion-binding site
The Strong ion-binding site from the BPN' subtilases can be transplanted into JP170 (or other subtilases in JP170 subgroup) by deletion of N79-N82 and subsequent insertion of LNNSIGV (SEQ ID NO:5), followed by the substitution A45D.N and optionally the substitutions E44P,T and/or R47Q.
Removal of ion-binding site in JP170
By removing an ion-binding site it is possible to decrease the enzymes dependency of calcium in the media. The ion-binding sites in JP170 or other JP170 type subtilases can be removed with guidance from information about the three-dimensional structures of other related subtilases, such as a BPN' type subtilase, such as Savinase or BPN', and a
TY145 type subtilase.
Removal of ion-binding site 1 can be done by deletion of N186-N199 and subse- quent insertion of at least three amino acid residues - or stated differently by the substitution of a region comprising from 3 to 6 amino acid residues for the region comprising 14 amino acid residues in positions 186 to 199, preferably the sequence of the substituting region is SSN (SEQ ID NO:6). Preferably, but not mandatory one or both of the substitutions I7Q and V3Y is further added. The ion-binding site 1 can be removed from a wild-type JP170 subtilase or a JP170 subtilase truncated as described above.
Subtilases free of ion-binding sites
Similarly, information about the three-dimensional structures of JP170 type subti- lases and TY145 type subtilases can be used to remove the Strong and Weak ion-binding sites in BPN' type subtilases, or the ion-binding sites in TY145 type subtilases may be removed on the b asis of structural i nformation a bout the J P170 and BPN' types of s ubtilases.
Using Savinase as an example, the removal can be done by altering the loops A194-L196 (weak ion-binding site) and L75-L82 (strong ion-binding site) either by a) insertion or deletion of a number of amino acid residues in the loops or b) by deletion of the entire loop or part of the loop and subsequent insertion of a number of residues from a corresponding loop of a JP17J0 or TY145 like subtilase.
Preferably the ion-binding sites of Savinase can be removed by either i) full or partial deletion of the region A194-L196 (BPN' numbers) and insertion of three or more residues chosen from JP170 positions P209-P217, and ii) full or partial deletion of the region L75-L82 (BPN' numbers) and insertion of at least one residue chosen from TY145 positions H83-Y92 or i) full or partial deletion of the region A194-L196 (BPN1 numbers) and insertion of three or more residues chosen from JP170 positions P209-P217 and ii) full or partial deletion of the region L75-L82 (BPN' numbers) and insertion of at least one residues chosen from JP170 positions N79-K83.
Removal of critical oxidation sites
In order to increase the stability of a JP170 type subtilase protease it may be advantageous to substitute or delete critical oxidation sites, such as methionines, with other amino acid residues which are not subject to oxidation.
Accordingly, in a further embodiment the present invention relates to an RP-II pro- tease variant, in which one or more amino acid residues susceptible to oxidation, especially m ethionine residues exposed to the surface of the molecule, is/are d eleted or replaced with another amino acid residue less susceptible to oxidation. The amino acid residue less susceptible to oxidation may for instance be selected from the group consisting of A, E, N, Q, I, L, S and K. Specific such variants comprises at least one of the deletions or substitutions
M42{*,S,A,N,Q,K}; M85{*,S,A,N,Q,K}; M97{*,S,A,N,Q,K}; M153f ,S,A,N,Q,K}
M220f,S,A,N,Q,K}; M250{*,S,A,N,Q,K}; and M255{*,S,A,N,Q,K} of the JP170 protease the deletions or substitutions M42{*,S,A,N,Q,K}; M85{*,S,A,N,Q,K}; M97{*,S,A,N,Q,K} M153f,S,A,N,Q,K}; M250f ,S,A,N,Q,K}; and M255f,S,A,N,Q,K} of the SD-521 and Ya proteases.
ϋ
Stabilization by modification of Asn-Gly pairs
It is known that at alkaline pH, the side chain of Asn may interact with the NH group of a sequential neighbouring amino acid to form an isoAsp residue where the backbone goes through the Asp side chain. This will leave the backbone more vulnerable to prote- olysis. The deamidation is much more likely to occur if the residue that follows is a Gly. Changing the Asn in front of the Gly or the Gly will prevent this from happening and thus improve the stability, especially as concerns thermo- and storage stability.
The invention consequently further relates to a subtilase, in which the modificatioins indicated above are either or both residues of any of the Asn-Gly sequence appearing in the amino acid sequence of the parent RP-II protease is/are deleted or substituted with a residue of a different amino acid.
The Asn and/or Gly residue may, for instance, be substituted with a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y.
More specifically, any of the Asn or Gly residues of the Asn-Gly occupying positions 66-67, 134-135 and/or 375-376 of the SD-521 and Ya protease; and positions 66-67, 134- 135, 301-302 and/or 375-376 of the JP170 protease, may be deleted or substituted with a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y. (positions are indicated in relation to the JP170 protease as indicated in Fig. 1 ).
Specific variants of JP170 are: N66f,A,Q,S,P,T,Y}; G67f ,A,Q,S,P,T,Y}; N134{*,A,Q,S,P,T,Y}; G135{*,A,Q,S,P,T,Y}; N301{*,A,Q,S,P,T,Y}; G302f ,A,Q,S,P,T,Y};
N375{*,A,Q,S,P,T,Y}; and G376{*,A,Q,S,P,T,Y}; and combinations thereof, such as
N66{*,A,Q,S,P,T,Y}+N134{*,A,Q,S,P,T,Y}, N66{*,A,Q,S,P,T,Y}+N301{*,A,Q,S,P,T,Y}, and
N66{*,A,Q,S,P,T,Y}+N375{*,A,Q,S,P,T,Y}, etc.
Specific variants of SD-521 and Ya proteases are: N66{*,A,Q,S,P,T,Y}; G67{*,A,Q,S,P,T,Y}; N134{*,A,Q,S,P,T,Y}; G135f ,A,Q,S,P,T,Y}; and N375{*,A,Q,S,P,T,Y}; G376{*,A,Q,S,P,T,Y}, and combinations thereof as indicated above.
Modification of Tyrosine residues
In relation to wash performance it has been found that the modification of certain tyro- sine residues to phenylalanine provides an improved wash performance. Without being bound by any specific theory, it is believed that titration of these Tyr residues in the alkaline wash liquor has negative effects that are alleviated by replacing the Tyr residues with other residues, especially Phe or Trp, particularly Phe.
In JP170 tyrosine residues may be modified in positions: 20, 54, 1 18, 137, 147, 194, 225, 247, 249, 334, 379, 388, 411 , and 418.
In SD-521 and Ya proteases the tyrosine residues may be modified in positions: 17, 20, 54, 137, 147, 187, 243, 247, 249, 299, 319, 334, 361 , 379, 386, 388, 411 , and 418.
In relation to JP170 the invention thus relates to the variants: Y17{F,W}, Y20{F,W},
Y54{F,W}, Y137{F,W}, Y147{F,W}, Y187{F,W}, Y243{F,W}, Y247{F,W}, Y249{F,W}, Y299{F,W}, Y319{F,W}, Y334{F,W}, Y361{F,W}, Y379{F,W}, Y386{F,W}, Y388{F,W},
Y411{F,W}, and Y418{F,W}. Corresponding modifications are easily identified in other
JP170 type subtilases.
Modification of tryptophan residues In order to stabilize the protein it may be advantageous to replace or delete tryptophan residues at the surface of the protein, e.g., as described in US 5,118,623. The tryptophan residues may advantageously be substituted for F, T, Q or G. Thus, in a further embodiment the invention relates to JP170 type subtilase variants comprising one or more of the following substitutions: For the SD-521 a nd Ya proteases positions 1 18, 1 29, 240, 306, 350, and 392; and for the JP170 protease positions 129, 240, 306, 350, and 392. Thus, the invention relates to a JP170 variant comprising one or more of the following substitutions W129{F,T,Q,G}, W240{F,T,Q,G}, W306{F,T,Q,G}, W350{F,T,Q,G}, and W392{F,T,Q,G}.
Combined modifications The present invention also encompasses any of the above mentioned subtilase variants in combination with any other modification to the amino acid sequence thereof. Especially combinations with other modifications known in the art to provide improved properties to the enzyme are envisaged.
Methods of preparing JP170 like or BPN' like subtilase variants
The subtilase variants, i.e. the JP170 and BPN' variants of the present invention may be produced by any known method within the art and the present invention also relates to nucleic acid encoding a subtilase variant of the present invention, a DNA construct comprising said nucleic acid and a host cell comprising said nucleic acid sequence. In general natural occurring proteins may be produced by culturing the organism expressing the protein and subsequently purifying the protein or it may be produced by cloning a nucleic acid, e.g. genomic DNA or cDNA, encoding the protein into an expression vector, introducing said expression vector into a host cell, culturing the host cell and purifying the expressed protein. Typically protein variants may be produced by site-directed mutagenesis of a parent protein, introduction into expression vector, host cell etc. The parent protein may be cloned from a strain producing the polypeptide or from an expression library, i.e. it may be isolated from genomic DNA or prepared from cDNA, or a combination thereof.
In general standard procedures for cloning of genes and/or introducing mutations (random and/or site directed) into said genes may be used in order to obtain a parent subtilase, or subtilase or subtilase variant of the invention. For further description of suitable techniques reference is made to Molecular cloning: A laboratory manual (Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.)); Current protocols in Molecular Biology (John Wiley and Sons, 1995; Harwood, C. R., and Cut- ting, S. M. (eds.)); Molecular Biological Methods for Bacillus (John Wiley and Sons, 1990); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleo- tide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds (1985)); Transcription And Translation (B.D. Hames & S.J. Higgins, eds. (1984)); Animal Cell Culture (R.I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); A Practical Guide To Molecular Cloning (B. Perbal, (1984)) and WO 96/34946. Further, variants could be constructed by:
Random Mutagenesis
Random mutagenesis is suitably performed either as localized or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid se- quence shown in question, or within the whole gene.
When the mutagenesis is performed by the use of an oligonucleotide, the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions that are to be changed. The doping or spiking may be done so that codons for unwanted amino acids are avoided. The doped or spiked oligonucleotide can be incorporated into the DNA encoding the subtilase enzyme by any published technique, using, e.g., PCR, LCR or any DNA polymerase and ligase as deemed appropriate.
Preferably, the doping is carried out using "constant random doping", in which the percentage of wild-type and modification in each position is predefined. Furthermore, the doping may be directed toward a preference for the introduction of certain nucleotides, and thereby a preference for the introduction of one or more specific amino acid residues. The doping may be made, e.g., so as to allow for the introduction of 90% wild type and 10% modifications in each position. An additional consideration in the choice of a doping scheme is based on genetic as well as protein-structural constraints. The doping scheme may be made by using the DOPE program which, inter alia, ensures that introduction of stop codons is avoided (L.J. Jensen et al. Nucleic Acid Research, 26, 697-702 (1998).
When PCR-generated mutagenesis is used, either a chemically treated or non- treated gene encoding a parent subtilase enzyme is subjected to PCR under conditions that increase the misincorporation of nucleotides (Deshler 1992; Leung et al., Technique, 1 , 1989, pp. 11-15).
The DNA sequence to be mutagenized may conveniently be present in a genomic or cDNA library prepared from an organism expressing the parent subtilase. Alternatively, the DNA sequence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or otherwise exposed to the mutagenising agent. The DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harbored in the cell. Fi- nally, the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence.
In some cases it may be convenient to amplify the mutated DNA sequence prior to performing the expression step b) or the screening step c). Such amplification may be performed in accordance with methods known in the art, the presently preferred method being PCR-generated amplification using oligonucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme.
The mutated DNA sequence may further comprise a DNA sequence encoding func- tions permitting expression of the mutated DNA sequence.
Localised random mutagenesis
The random mutagenesis may be advantageously localised to a part of the parent subtilase i n q uestion. This may, e .g., be advantageous when certain regions of the e n- zyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.
The localised or region-specific, random mutagenesis is conveniently performed by use of PCR generated mutagenesis techniques as described above or any other suitable technique known in the art. Alternatively, the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g., by insertion into a suitable vector, and said part m ay be subsequently subjected to m utagenesis by use of any of the m utagenesis methods discussed above.
General method for random mutagenesis by use of the DOPE program The random mutagenesis may be carried out by the following steps:
1. Select regions of interest for modification in the parent enzyme
2. Decide on mutation sites and non-mutated sites in the selected region 3. Decide on which kind of mutations should be carried out, e.g. with respect to the desired stability and/or performance of the variant to be constructed
4. Select structurally reasonable mutations
5. Adjust the residues selected by step 3 with regard to step 4.
6. Analyse by use of a suitable dope algorithm the nucleotide distribution. 7. If necessary, adjust the wanted residues to genetic code realism, e.g. taking into account constraints resulting from the genetic code, e.g. in order to avoid introduc- tion of stop codons; the skilled person will be aware that some codon combinations cannot be used in practice and will need to be adapted
8. Make primers
9. Perform random mutagenesis by use of the primers
5 10. Select resulting subtilase variants by screening for the desired improved properties.
Suitable dope algorithms for use in step 6 are well known in the art. One such algorithm is described by Tomandl, D. et al., 1997, Journal of Computer-Aided Molecular Design 11 :29-38. Another algorithm is DOPE (Jensen, LJ, Andersen, KV, Svendsen, A, and o Kretzschmar, T (1998) Nucleic Acids Research 26:697-702).
Expression vectors
A recombinant expression vector comprising a nucleic acid sequence encoding a subtilase variant of the invention may be any vector that may conveniently be subjected to 5 recombinant DNA p rocedures a nd which may bring about the expression of the nucleic acid sequence.
The choice of vector will often depend on the host cell into which it is to be intro- duced. Examples of a suitable vector include a linear or closed circular plasmid or a virus. The vector may be an autonomously replicating vector, i.e., a vector which exists as an 0 extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, pACYC184, pUB110, pE194, pTA1060, and pAMβl . Examples of origin of 5 replications for use in a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARS1. The origin of replication may be one having a mutation which makes it function as temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433). 0 Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Vectors which are integrated into the genome of the host cell may contain any nucleic acid sequence enabling integration into the genome, in particular it may contain nucleic acid sequences facilitating integration into the genome by homolo- 5 gous or non-homologous recombination. The vector system may be a single vector, e.g. plasmid or virus, or two or more vectors, e.g. plasmids or virus', which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
The vector may in particular be an expression vector in which the DNA sequence encoding the subtilase variant of the invention is operably linked to additional segments or control sequences required for transcription of the DNA. The term, "operably linked" indi- cates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence encoding the subtilase variant. Additional segments or control sequences include a promoter, a leader, a polyadenylation sequence, a propeptide sequence, a signal sequence and a transcription terminator. At a minimum the control sequences include a pro- moter and transcriptional and translational stop signals.
The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
Examples of suitable promoters for use in bacterial host cells include the promoter of the Bacillus subtilis levansucrase gene (sacB), the Bacillus stearothermophilus malto- genic amylase gene (amyM), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, o
Proceedings of the National Academy of Sciences USA 75:3727-3731). Other examples include the phage Lambda PR or PL promoters or the E. coli lac, trp or tac promoters or the Streptomyces coelicolor agarase gene (dagA). Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and in Sam- brook et al., 1989, supra.
Examples of suitable promoters for use in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhi- zomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae those phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (as described in U.S. Patent No. 4,288,627, which is incorporated herein by reference), and hybrids thereof. Particularly preferred promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes en- coding Aspergillus niger neutral (-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters. Further suitable promoters for use in filamentous fungus host cells are the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093 - 2099) or the tpiA promoter.
Examples of suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419 - 434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPM (US 4,599,311 ) or ADH2-4c (Russell et al., Nature 304 (1983), 652 - 654) promoters.
Further useful promoters are obtained from the Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae galactokinase gene (GAL1 ), the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488. In a mammalian host cell, useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).
Examples of suitable promoters for use in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981 ), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814) or the adenovirus 2 major late promoter.
An example of a suitable promoter for use in insect cells is the polyhedrin promoter (US 4,745,051 ; Vasuvedan et al., FEBS Lett. 311 , (1992) 7 - 11), the P10 promoter (J.M. Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica polyhedro- sis virus basic protein promoter (EP 397 485), the baculovirus immediate early gene 1 promoter (US 5,155,037; US 5,162,222), or the baculovirus 39K delayed-early gene promoter (US 5,155,037; US 5,162,222).
The DNA sequence encoding a subtilase variant of the invention may also, if necessary, be operably connected to a suitable terminator.
The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like ampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, neomycin, hygromycin, methotrexate, or resistance to heavy metals, virus or herbicides, or which provides for prototrophy or auxotrophs. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, resistance. A frequently used mammalian marker is the dihydrofolate reductase gene (DHFR). Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3. A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate d ecarboxylase), sC ( sulfate a denyltransferase), trpC (anthranilate synthase), and g lufosinate resistance markers, as well as equivalents from other species. Particularly, for use in an Aspergillus cell are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus. Furthermore, selection may be accomplished by co-transformation, e.g., as described in WO 91/17243, where the selectable marker is on a separate vector.
To direct a subtilase variant of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the enzyme in the correct reading frame. Secretory signal sequences are commonly positioned 5* to the DNA sequence encoding the enzyme. The secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein.
The procedures used to ligate the DNA sequences coding for the present enzyme, the promoter and optionally the terminator and/or secretory signal sequence, respectively, or to assemble these sequences by suitable PCR amplification schemes, and to insert them into suitable vectors containing the information necessary for replication or integration, are well known to persons skilled in the art (cf., for instance, Sambrook et al.).
More than one copy of a nucleic acid sequence encoding an enzyme of the present invention may be inserted into the host cell to amplify expression of the nucleic acid sequence. Stable amplification of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome using methods well known in the art and selecting for transformants.
The nucleic acid constructs of the present invention may also comprise one or more nucleic acid sequences which encode one or more factors that are advantageous in the expression of the polypeptide, e.g., an activator (e.g., a trans-acting factor), a chaperone, and a processing protease. Any factor that is functional in the host cell of choice may be used in the present invention. The nucleic acids encoding one or more of these factors are not necessarily in tandem with the nucleic acid sequence encoding the polypeptide.
Host cells The DNA sequence encoding a subtilase variant of the present invention may be either homologous or heterologous to the host cell into which it is introduced. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal se- quence and/or terminator sequence than in its natural environment. The term "homologous" is intended to include a DNA sequence encoding an enzyme native to the host organism in question. The term "heterologous" is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence. The host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell that is capable of producing the present subtilase variants, such as prokaryotes, e.g. bacteria or eukaryotes, such as fungal cells, e.g. yeasts or filamentous fungi, insect cells, plant cells or mammalian cells.
Examples of bacterial host cells which, on cultivation, are capable of producing the subtilase variants of the invention are gram-positive bacteria such as strains of Bacillus, e.g. strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gram- negative bacteria such as Escherichia coli or Pseudomonas sp. The transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
When expressing the subtilase variant in bacteria such as E. coli, the enzyme may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or it may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the enzyme is refolded by diluting the denaturing agent. In the latter case, the enzyme may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the enzyme. When expressing the subtilase variant in gram-positive bacteria such as Bacillus or
Streptomyces strains, the enzyme may be retained in the cytoplasm, or it may be directed to the extracellular medium by a bacterial secretion sequence. In the latter case, the enzyme may be recovered from the medium as described below.
Examples of host yeast cells include cells of a species of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, Hansehula, or Yarrowia. In a p articular embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cer- evisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. Other useful yeast host cells are a Kluyveromyces lactis, Kluyveromyces fragilis, Hansehula polymorpha, Pichia pastoris, Yarrowia lipolytica, Schizosaccharomyces pombe, Ustilgo maylis, Candida maltose, Pichia guillermondii and Pichia methanolio cell (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; US 4,882,279 and US 4,879,231). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980. The biology of yeast and manipulation of yeast genetics are well known in the art (see, e.g., Biochemistry and Genetics of Yeast, Bacil, M.,MHorecker, B.J., and Stopani, A.O.M., editors, 2nd edition, 1987; The Yeasts, Rose, A.H., and Harrison, J.S., editors, 2nd edition, 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathern et al., editors, 1981). Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzy- mology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75:1920.
Examples of filamentous fungal cells include filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra), in particular it may of the a cell of a species of Acremonium, such as A. chrysogenum, Aspergillus, such as A. awamori, A. foetidus, A. japonicus, A. niger, A. nidulans or A. oryzae, Fusarium, such as F. bactridioides, F. cerealis, F. crookwellense, F. culmorum, F. graminearum, F. graminum, F. heterosporum, F. negundi, F. reticulatum, F. roseum, F. sambucinum, F. sarcochroum, F. sulphureum, F. trichothecioides or F. oxysporum, Humicola, such as H. insolens or H. lanuginose, Mucor, such as M. miehei, Myceliophthora, such as M. thermo- philum, Neurospora, such as N. crassa, Penicillium, such as P. purpurogenum, Thielavia, such as T. terrestris, Tolypocladium, or Trichoderma, such as T. harzianum, T. koningii, T. longibrachiatum, T. reesei or T. viride, or a teleomorph or synonym thereof. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 230 023.
Examples of insect cells include a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. US 5,077,214). Culture conditions may suitably be as described in WO 89/01029 or WO 89/01028.Transformation of insect cells and pro- duction of heterologous polypeptides therein may be performed as described in US 4,745,051 ; US 4, 775, 624; US 4,879,236; US 5,155,037; US 5,162,222; EP 397,485). Examples of mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection. Methods of transfect- ing mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601 - 621 ; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327 - 341 ; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422 - 426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981 ), 603, Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., N.Y., 1987, Hawley-Nelson et al., Focus 15 (1993), 73; Ciccarone et al., Focus 15 (1993), 80; Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841 - 845. Mammalian cells may be transfected by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52:546).
Methods for expression and isolation of proteins To express an enzyme of the present invention the above mentioned host cells transformed or transfected with a vector comprising a nucleic acid sequence encoding an enzyme of the present invention are typically cultured in a suitable nutrient medium under conditions permitting the production of the desired molecules, after which these are recovered from the cells, or the culture broth. The medium used to culture the host cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The media may be prepared using procedures known in the art (see, e.g., references o for bacteria and yeast; Bennett, J.W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).
If the enzymes of the present invention are secreted into the nutrient medium, they may be recovered directly from the medium. If they are not secreted, they may be recovered from cell lysates. The enzymes of the present invention may be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the enzyme in question. The enzymes of the invention may be detected using methods known in the art that are specific for these proteins. These detection methods include use of specific antibodies, formation of a product, or disappearance of a substrate. For example, an enzyme assay may be used to determine the activity of the molecule. Procedures for determining various kinds of activity are known in the art.
The enzymes of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J-C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). When an expression vector comprising a DNA sequence encoding an enzyme of the present invention is transformed/transfected into a heterologous host cell it is possible to enable heterologous recombinant production of the enzyme. An advantage of using a heterologous host cell is that it is possible to make a highly purified enzyme composition, characterized in being free from homologous impurities, which are often present when a protein or peptide is expressed in a homologous host cell. In this context homologous impurities mean any impurity (e.g. other polypeptides than the enzyme of the invention) which originates from the homologous cell where the enzyme of the invention is originally obtained from.
DETERGENT APPLICATIONS
The enzyme of the invention may be added to and thus become a component of a detergent composition.
The d etergent composition of the i nvention m ay for example be formulated a s a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations, especially for automatic dish washing (ADW).
In a specific aspect, the invention provides a detergent additive comprising the en- zyme of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galac- tanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.
In general the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non- enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts. Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Mi- crobial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metallo protease, preferably an alkaline mi- crobial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carisberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583. Examples of useful proteases are the variants described in WO 92/19729, WO
98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101 , 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.
Preferred commercially available protease enzymes include Alcalase™, Savi- nase™, Primase™, Duralase™, Esperase™, and Kannase™ (Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™, FN2™, and FN3™ (Genencor International Inc.).
Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseu- domonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. ce- pacia (EP 331 376), P. stutzeri (GB 1 ,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131 , 253-360), B. stearothermophilus (JP 64/744992) or β. pumilus (WO 91/16422).
Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541 , EP 407 225, EP 260 1 05, WO 95/35381 , WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.
Preferred commercially available lipase enzymes include Lipolase™ and Lipolase Ultra™ (Novozymes A/S).
Amylases: Suitable amylases (α and/or β) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, α-amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, de- scribed in more detail in GB 1 ,296,839.
Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391 , 408, and 444.
Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ and BAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from Genencor International Inc.).
Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modi- fied or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259. Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299. Commercially available cellulases include Celluzyme™, and Carezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor International Inc.), and KAC- 500(B)™ (Kao Corporation).
Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include Guardzyme™ (Novozymes A/S).
The detergent enzyme(s) may be included i n a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granu- lates, liquids, in particular stabilized liquids, or slurries.
Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 30000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Pro- tected enzymes may be prepared according to the method disclosed in EP 238,216.
The detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or non-aqueous.
The detergent composition comprises one or more surfactants, which may be non- ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight.
When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, al- pha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
When included therein the detergent will usually contain from about 0.2% to about
40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkyl- polyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine ("glucamides").
The detergent may contain 0-65 % of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic a cid, d iethylenetriaminepentaacetic a cid, a Ikyl- o r a Ikenylsuc- cinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
The detergent may comprise one or more polymers. Examples are carboxymethyl- cellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylat.es such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
The detergent may contain a bleaching system which may comprise a H202 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type. The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708. The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners i ncluding clays, foam boosters, suds s uppressors, a nti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
In the detergent compositions any enzyme, in particular the enzyme of the inven- tion, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per litre of wash liquor, preferably 0.05-5 mg of enzyme protein per litre of wash liquor, in particular 0.1-1 mg of enzyme protein per litre of wash liquor.
The enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.
MATERIALS AND METHODS
Textiles
Standard textile pieces are obtained from EMPA St. Gallen, Lerchfeldstrasse 5, CH-9014 St. Gallen, Switzerland. Especially type EMPA 116 (cotton textile stained with blood, milk and ink) and EMPA 117 (polyester/cotton textile stained with blood, milk and ink). Other atandard textile pieces are obtained from wfk-Cleaning Technology Research Institute, Chris- tenfeld 10, D-41379 Brϋggen-Bracht, Germany. Especially type wfk10N (cotton textile stained with egg/pigment), wfklOeggEG (cotton textile stained with egg yolk). Denaturation of wfk10N occurs in an autoclave.
Method for producing a subtilase variant
The present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture. When an expression vector comprising a DNA sequence encoding the enzyme is transformed into a heterologous host cell it is possible to enable heterologous recombinant production of the enzyme of the invention. Thereby it is possible to make a highly purified subtilase composition, characterized in being free from homologous impurities. The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question. The expressed subtilase may conveniently be secreted into the culture medium and may be recovered there-from by well- known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
EXAMPLE 1
Removal of ion-binding sites from BPN' like subtilases The below mentioned regions in JP170 and TY145 have been selected for transfer from JP170 and TY145 to Savinase. By use of the molecular methods of preparing subtilase variants as described herein, the Savinase regions (BPN' numbering) are deleted and the JP170 and TY145 regions are inserted instead. Since the Savinase regions are in contact with ion-binding sites,, the purpose of the modifications is to remove the ion-binding site from Savinase.
Savinase region A194-L196
JP170 region P209-P217 and
Savinase region L75-L82 TY145 region H83-Y92,
alternatively the modification can be
Savinase region A194-L196 JP170 region P209-P217 and
Savinase region L75-L82
JP170 region N79-K83.
Construction and expression of enzyme variants: Site-directed mutagenesis:
Subtilase JP170 site-directed variants of the invention comprising specific inser- tions/deletions/substitutions are made by traditional cloning of DNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) produced by PCR with oligos containing the desired mutations.
The template plasmid DNA may be pSX222, or an analogue of this containing a variant of subtilisin JP170. Mutations are introduced by oligo directed mutagenesis to the construction of variants.
The subtilisin JP170 variants were transformed into E. coli. DNA purified from an over night culture of these transformants is transformed into B. subtilis by restriction endonuclease digestion, purification of DNA fragments, ligation, transformation of S. subtilis. Transformation of B. subtilis i s performed as described by Dubnau et al., 1971 , J. M ol. Biol. 56, pp. 209-221.
Site-directed mutagenesis in order to introduce mutations in a specific region: The overall strategy used to perform site-directed mutagenesis is: Mutagenic primers (oligonucleotides) are synthesized corresponding to the DNA sequence flanking the sites of mutation, separated by the DNA base pairs defining the insertions / deletions / substitutions.
Subsequently, the resulting mutagenic primers are used in a PCR reaction with the modified plasmid pSX222. The resulting PCR fragment is purified and extended in a sec- ond PCR-reaction, the resulting PCR product is purified and extended in a third PCR- reaction before being digegted by endonucleases and cloned into the E. coli - B. subtilis shuttle vector pSX222. The PCR reactions are performed under normal conditions. The plasmid DNA is transformed into E. coli by well-known techniques and one E. coli colony is sequenced to confirm the mutation designed. In order to purify subtilase variants of the invention, the pSX222 expression plasmid comprising a variant of the invention was transformed into a competent B. subtilis strain and fermented as described above.
EXAMPLE 2 Purification and assessment of enzyme concentration
After fermentation, purification of subtilisin variants is accomplished u sing H ydro- phobic Charge Induction Chromatography (HCIC) and subsequent vacuum filtration. To capture the enzyme, the HCIC uses a cellulose matrix to which 4-Mercapto-Ethyl- Pyridine (4-MEP) is bound. Beads of the cellulose matrix sized 80-100 μm are mixed with a media containing yeast and the transformed B. subtilis capable of secreting the subtilisin variants and incu- bated at pH 9.5 in Unifilter® microplates.
As 4-MEP is hydrophobic at pH > 7 and the subtilisin variants are hydrophobic at pH 9.5 a hydrophobic association is made between the secreted enzyme and the 4-MEP on the beads. After incubation the media and cell debris is removed by vacuum filtration while the beads and enzyme are kept on the filter.
To elute the enzyme from the beads the pH is now lowered by washing the filter with a n elution b uffer ( pH 5 ) H ereby the e nzymes part from the beads a nd can be retrieved from the buffer.
The concentration of the purified subtilisin enzyme variants is assessed by active site titration (AST).
The purified enzyme is incubated with the high affinity inhibitor CI-2A at different concentrations to inhibit a varying amount of the active sites. The protease and inhibitor o binds to each other at a 1 :1 ratio and accordingly the enzyme concentration can be directly related to the concentration of inhibitor, at which all protease is inactive. To measure the residual protease activity, a substrate (0.6 mM Suc-Ala-Ala-Pro-Phe-pNA in Tris/HCI buffer) is added after the incubation with inhibitor and during the following 4 minutes the development of the degradation product pNA (paranitrophenol) is measured periodically at
405 nm on an Elisa Reader.
Each of the variants listed below were constructed as described above. H243K; S238K; L233T; L233S; L233D; Y247R; H200D; H200A; H200G; E185D; S193Q;
S193Y; N390D; G394N; G394F; W240H; G355A; G355S; N316D; N79D; K246R; K83R;
H200D+D196N; H243E.
EXAMPLE 3 Automatic Mechanical Stress Assay (AMSA).
Description of AMSA-test method:
Washing experiments are performed in order to asses the wash performance of selected JP170 subtilase vgπants in detergent compositions. Subtilases of the present application were tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA, the wash performance of a large quantity of small volume enzyme-detergent solutions can be examined The AMSA plate has a number of slots for test solutions and a lid firmly squeezing the textile swatch to be washed against all the slot openings. During the washing time, the plate, test solutions, textile and lid are vigorously shaken to bring the test solution in contact with the textile and apply mechanical stress in a regular, periodic oscillating manner. For further description see WO 02/42740 especially the paragraph "Special method embodiments" at page 23-24. The experiment was conducted under the experimental conditions specified below:
Commercial detergent base European 3in1 ADW type
Detergent dosage 5 - 5.5 g/L
Test solution volume 160 μL
PH As is
Wash time 20 minutes
Temperature 50°C
Water hardness 25°dH
0.25mg/L, 0.5mg/L, 1mg/L, and
Enzyme concentration in test solution 2.,5 mg/L for wfk10N;
1mg/L, 2,5mg/L, 4mg/L, and 6mg/L for denatured wfk10N.
Test material Wfk10N
Water hardness was adjusted to 9°dH by addition of CaCI2, MgCI2, and NaHC03 (Ca2+:Mg2+ = 4:1) to the test system. After washing the textile pieces were flushed in tap water and dried.
The performance of the enzyme variant is measured as the brightness of the colour of the textile samples washed with that specific protease. Brightness can also be expressed as the intensity of the light reflected from the textile sample when illuminated with white light. When the textile is stained the intensity of the reflected light is lower, than that of a c lean t extile. T herefore t he i ntensity o f t he r eflected I ight c an be u sed t o m easure wash performance of a shuffled protease.
Colour measurements are made with a professional flatbed scanner (PFU DL2400pro, obtainable from: J.M. Thomsen, Dorfgade 2, Dorf, Dronninglund, DK-9330), which is used to capture an image of the washed textile samples. The scans are made with a resolution of 200 dpi and with an output colour dept of 24 bits. In order to get accurate results, the scanner is frequently calibrated with a Kodak reflective IT8 target.
To extract a value for the l ight i ntensity f rom the scanned i mages, a special d esigned software application is used {Novozymes Color Vector Analyzer). The program re- trieves the 24 bit pixel values from the image and converts them into values for red, green and blue (RGB). The intensity value (Int) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:
Detergents
Detergents for wash performance tests of the subtilases of the invention can be ob- tained by purchasing fully formulated commercial detergents at the market and subsequently inactivate the enzymatic components by heat treatment (5 minutes at 85°C in aqueous solution). Moreover a commercial detergent base without enzymes can be purchased directly from the manufacturer. Further a suitable model detergent can be purchased and used for wash performance tests. The proteases may also be tested in a model detergent composition comprising
Sodium Tripolyphosphate 23.0%
Sodium Citrate Dihydrate 22.3%
Sodium Perborate MonohyαYate 6.0%
Tetraacetyl Ethylendiamine 2.0% Sodium Disilicate (noncrystaline) 5.0%
Linear Fatty Alcohol Ethoxylate 2.0%
(non-ionic surfactant, low foaming) Maleic acid/Acrylic acid copolymer 4.0%
(Sodium salt, 50% active on Sodium Carbonate) Sodium Carbonate, anhydrous add to 100%
Using the above test method in combination with a commercially available detergent the results shown below were obtained. As it appears, the subtilases according to the invention e xhibits i mproved wash p erformance o n e gg s tains i n c omparison t o t he wiild type JP170 subtilase with SEQ ID NO:1.
APPENDIX 1
REMARK Complex of JP170 and CI2A inhibitor REMARK Contents of asymmetric unit subtilisin 2x (433 a. a. x 2) REMARK CI2A inhibitor 2x (a. a. 16 - 83 and 21 - 83) REMARK small peptide (autodigestion product, a. a. KPSLL, 280 - 284) REMARK Ca ions 6x, H20 1115 x REMARK REMARK Crystallization conditions: (AMB) Hanging drop vapour diffusion REMARK method where the drop consists of 2 μl of 15 - 20 mg.ml-1 REMARK protein concentration, 10 mM Na cacodylate - HC1 buffer, pH 6.5 REMARK and 1 μl of the well solution, 20% w/v PEG 4000, 0.1 M Hepes REMARK buffer, pH 7.5, 10% v/v isopropanol. HEADER XX-XXX-XX xxxx COMPND REMARK REMARK REFINEMENT . REMARK PROGRAM REFMAC 5.1.24 REMARK AUTHORS MURSHUDOV, VAGIN , DODSON REMARK REMARK REFINEMENT TARGET MAXIMUM LIKELIHOOD REMARK REMARK DATA USED IN REFINEMENT. REMARK RESOLUTION RANGE HIGH (ANGSTROMS) 1.90 REMARK RESOLUTION RANGE LOW (ANGSTROMS) 19.96 REMARK DATA CUTOFF (SIGMA (F) ) NONE REMARK COMPLETENESS FOR RANGE (%) 76.65 REMARK NUMBER OF REFLECTIONS 59444 REMARK REMARK FIT TO DATA USED IN REFINEMENT. REMARK CROSS-VALIDATION METHOD NULL REMARK FREE R VALUE TEST SET SELECTION NULL REMARK R VALUE (WORKING + TEST SET) 0.12256 REMARK R VALUE (WORKING SET) 0.12256 REMARK FREE R VALUE NULL REMARK FREE R VALUE TEST SET SIZE (%) NULL REMARK FREE R VALUE TEST SET COUNT NULL REMARK REMARK FIT IN THE HIGHEST RESOLUTION BIN. REMARK TOTAL NUMBER OF BINS USED 20 REMARK BIN RESOLUTION RANGE HIGH 1.901 REMARK BIN RESOLUTION RANGE LOW 1.950 REMARK REFLECTION IN BIN (WORKING SET) 940 REMARK BIN R VALUE (WORKING SET) 0.149 REMARK BIN FREE R VALUE SET COUNT 0 REMARK BIN FREE R VALUE -999.000 REMARK REMARK NUMBER OF NON -HYDROGEN ATOMS USED IN REFINEMENT. REMARK ALL ATOMS 8694 REMARK REMARK B VALUES. REMARK FROM WILSON PLOT (A**2) NULL REMARK MEAN B VALUE (OVERALL, A**2) 16.479 REMARK OVERALL ANISOTROPIC B VALUE. REMARK Bll (A**2) 0.05 REMARK B22 (A**2) 0.06 REMARK B33 (A**2) -0.11 REMARK B12 (A**2) 0.00 REMARK B13 (A**2) 0.00 REMARK B23 (A**2) 0.00 REMARK REMARK ESTIMATED OVERALL COORDINATE ERROR. REMARK ESU BASED ON R VALUE (A) 0.151 REMARK 3 ESU BASED ON FREE R VALUE (A) NULL REMARK 3 ESU BASED ON MAXIMUM LIKELIHOOD (A) 0.052 REMARK 3 ESU FOR B VALUES BASED ON MAXIMUM LIKELIHOOD (A**2) 1.828 REMARK 3 REMARK 3 CORRELATION COEFFICIENTS. REMARK 3 CORRELATION COEFFICIENT FO-FC 0.969 REMARK 3 CORRELATION COEFFICIENT FO-FC FREE NULL REMARK 3 REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES COUNT RMS WEIGHT REMARK 3 BOND LENGTHS REFINED ATOMS (A) 7733 0.014 0 . 021 REMARK 3 BOND LENGTHS OTHERS (A) 6857 0.001 0 . 020 REMARK 3 BOND ANGLES REFINED ATOMS (DEGREES) 10540 1.478 1 . 936 REMARK 3 BOND ANGLES OTHERS (DEGREES) 15972 0.815 3 . 000 REMARK 3 TORSION ANGLES, PERIOD 1 (DEGREES) 997 15.784 5 . 000 REMARK 3 CHIRAL-CENTER RESTRAINTS (A**3) 1197 0.106 0 . 200 REMARK 3 GENERAL PLANES REFINED ATOMS (A) 8819 0.007 0 . 020 REMARK 3 GENERAL PLANES OTHERS (A) 1500 0.008 0 . 020 REMARK 3 NON-BONDED CONTACTS REFINED ATOMS (A) 1552 0.221 0 . 300 REMARK 3 NON-BONDED CONTACTS OTHERS (A) 8282 0.265 0 . 300 REMARK 3 NON-BONDED TORSION OTHERS (A) 4417 0.089 0 . 500 REMARK 3 H-BOND (X...Y) REFINED ATOMS (A) 1391 0.198 0 . 500 REMARK 3 POTENTIAL METAL- ION REFINED ATOMS (A) 25 0.145 0 . 500 REMARK 3 SYMMETRY VDW REFINED ATOMS (A) 10 0.129 0 . 300 REMARK 3 SYMMETRY VDW OTHERS (A) 57 0.268 0 . 300 REMARK 3 SYMMETRY H-BOND REFINED ATOMS (A) 87 0.272 0 . 500 REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS. COUNT RMS WEIGHT REMARK 3 MAIN-CHAIN BOND REFINED ATOMS (A**2) 4985 ; 0.697 1.500 REMARK 3 MAIN-CHAIN ANGLE REFINED ATOMS (A**2) 8031 ; 1.205 2.000 REMARK 3 SIDE-CHAIN BOND REFINED ATOMS (A**2) 2746 ; 1.963 3.000 REMARK 3 SIDE-CHAIN ANGLE REFINED ATOMS (A**2) 2509 ; 3.180 4.500 REMARK 3 REMARK 3 NCS RESTRAINTS STATISTICS REMARK 3 NUMBER OF NCS GROUPS : NULL REMARK 3 REMARK 3 REMARK 3 TLS DETAILS REMARK 3 NUMBER OF TLS GROUPS NULL REMARK 3 REMARK 3 REMARK 3 BULK SOLVENT MODELLING. REMARK 3 METHOD USED : BABINET MODEL WITH MASK REMARK 3 PARAMETERS FOR MASK CALCULATION REMARK 3 VDW PROBE RADIUS 1.40 REMARK 3 ION PROBE RADIUS 0.80 REMARK 3 SHRINKAGE RADIUS 0.80 REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: REMARK 3 HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS REMARK 3 CISPEP 1 GLY A 163 PRO A 164 00 CISPEP 2 ALA A 171 PRO A 172 00 CISPEP 3 PHE A 191 GLY A 192 00 CISPEP 4 ASN A 199 HIS A 200 00 CISPEP 5 GLY A 208 PRO A 209 00 CISPEP 6 LYS A 216 PRO A 217 00 CISPEP 7 ASP A 236 SER A 237 00 CISPEP 8 ASP A 244 SER A 245 00 CISPEP 9 PHE A 299 PRO A 300 00 CISPEP 10 SER A 327 THR A 328 00 CISPEP 11 ALA A 386 PRO A 387 00 CISPEP 12 GLU A 414 VAL A 415 00 CISPEP 13 GLY A 423 PRO A 424 00 LINK ASN B 316 LYS B 318 gap
LINK GLU B 330 ALA B 332 gap
LINK LEU B 337 LYS B 340 gap
LINK GLU D 330 ALA D 332 gap
LINK LEU D 337 LYS D 340 gap
CISPEP 14 GLY C 163 PRO C 164 0.00
CISPEP 15 ALA C 171 PRO C 172 0.00
CISPEP 16 PHE C 191 GLY c 192 0.00
CISPEP 17 ASN C 199 HIS c 200 0.00
CISPEP 18 GLY C 208 PRO c 209 0.00
CISPEP 19 LYS C 216 PRO c 217 0.00
CISPEP 20 ASP C 236 SER c 237 0.00
CISPEP 21 ASP C 244 SER c 245 0.00
CISPEP 22 PHE C 299 PRO c 300 0.00
CISPEP 23 SER C 327 THR c 328 0.00
CISPEP 24 ALA C 386 PRO c 387 0.00
CISPEP 25 GLU C 414 VAL c 415 0.00
CISPEP 26 GLY C 423 PRO c 424 0.00
CRYST1 58. .387 151.411 64.054 90.00 117.11 90.00 P 1 21 1
SCALE1 0.017127 0 .000000 0.008768 0.00000
SCALE2 0.000000 0 .006605 0.000000 0.00000
SCALE3 0.000000 0 .000000 0.017539 0.00000
HETATM 1 N ASN A 1 18.066 20 .808 -3.996 1.00 14 .87 A N
HETATM 2 C9 ASN A 1 18.461 22 .053 -3.689 1.00 14 .47 A c
HETATM 3 O10 ASN A 1 19.168 22 .251 -2.661 1.00 13 .33 A O
HETATM 4 Oil ASN A 1 18.108 23 .029 -4.423 1.00 14 .69 A O
HETATM 5 CA ASN A 1 18.499 19 .635 -3.189 1.00 14 .35 A c
HETATM 6 CB ASN A 1 18.164 18 .329 -3.883 1.00 14 .69 A c
HETATM 7 CG ASN A 1 16.670 18 .063 -4.031 1.00 14 .08 A c
HETATM 8 ND2 ASN A 1 16.271 17 .100 -5.019 1.00 12 .20 A N
HETATM 9 OD1 ASN A 1 15.768 18 .701 -3.206 1.00 14 .76 A O
HETATM 10 C ASN A 1 19.990 19 .659 -2.890 1.00 14 .84 A c
HETATM 11 O ASN A 1 20.353 19 .313 ■ -1.601 1.00 14 .20 A O
ATOM 12 N ASP A 2 20.881 19 .935 -3.834 1.00 15 .84 A N
ATOM 13 CA ASP A 2 22.306 19 .835 -3.520 1.00 16 .82 A c
ATOM 14 CB ASP A 2 23.178 20 .088 -4.763 1.00 17 .53 A C
ATOM 15 CG ASP A 2 23.121 18 .947 -5.783 1.00 18 .18 A C
ATOM 16 OD1 ASP A 2 22.652 17 .811 -5.493 1.00 20 .58 A O
ATOM 17 OD2 ASP A 2 23.544 19 .106 -6.931 1.00 22 .02 A O
ATOM 18 C ASP A 2 22.712 20 .816 -2.413 1.00 17 .23 A C
ATOM 19 O ASP A 2 23.671 20 .562 -1.703 1.00 18 .17 A o
ATOM 20 N VAL A 3 22.018 21 .952 -2.304 1.00 17 .05 A N
ATOM 21 CA VAL A 3 22.374 22 .945 -1.311 1.00 16 .07 A C
ATOM 22 CB VAL A 3 21.974 24 .356 -1.701 1.00 16 .60 A C
ATOM 23 CGI VAL A 3 22.327 25 .323 -0.560 1.00 16 .25 A C
ATOM 24 CG2 VAL A 3 22.676 24 .770 -3.003 1.00 18 .81 A C
ATOM 25 C VAL A 3 21.749 22 .565 0.033 1.00 15 .65 A C
ATOM 26 O VAL A 3 22.431 22 .603 1.090 1.00 14 .41 A O
ATOM 27 N ALA A 4 20.497 22 .119 -0.012 1.00 13 .75 A N
ATOM 28 CA ALA A 4 19.824 21 .664 1.196 1.00 14 .03 A C
ATOM 29 CB ALA A 4 18.388 21 .260 0.881 1.00 13 .78 A C
ATOM 30 C ALA A 4 20.544 20 .512 1.876 1.00 14 .28 A C
ATOM 31 O ALA A 4 20.548 20 .406 3.110 1.00 14 .07 A O
ATOM 32 N ARG A 5 21.093 19 .617 1.064 1.00 13 .74 A N
ATOM 33 CA ARG A 5 21.807 18, .445 1.553 1.00 14 .95 A C
ATOM 34 CB ARG A 5 22.395 17, .709 0.349 1.00 15, .61 A C
ATOM 35 CG ARG A 5 23.452 16, .639 0.631 1.00 17 .28 A C
ATOM 36 CD ARG A 5 23.873 15, .945 -0.672 1.00 20 .73 A C
ATOM 37 NE ARG A 5 24.802 14, .852 -0.459 1.00 21, .95 A N
ATOM 38 CZ ARG A 5 26.128 14, .986 -0.513 1.00 24 .69 A C
ATOM 39 NH1 ARG A 5 26.687 16, .173 -0.793 1.00 25 .62 A N
ATOM 40 NH2 ARG A 5 26.898 13 .933 -0.290 1.00 22 .96 A N
ATOM 41 C ARG A 5 22.918 18 .840 2.515 1.00 14 .83 A C ATOM 42 O ARG A 5 23.135 18.195 3.546 1.00 14.86 A O
ATOM 43 N GLY A 6 23 .641 19 .897 2 .166 1 .00 15 .33 A N
ATOM 44 CA GLY A 6 24 .677 20 .416 3 .044 1 .00 15 .34 A C
ATOM 45 C GLY A 6 24 .094 21 .124 4 .257 1 .00 15 .01 A C
ATOM 46 O GLY A 6 24 .609 20 .980 5 .362 1 .00 14 .62 A O
ATOM 47 N ILE A 7 23 .018 21 .879 4 .062 1 .00 14 .44 A N
ATOM 48 CA ILE A 7 22 .411 22 .613 5 .168 1 .00 13 .98 A C
ATOM 49 CB ILE A 7 21 .266 23 .505 4 .698 1 .00 13 .68 A C
ATOM 50 CGI ILE A 7 21 .813 24 .676 3 .864 1 .00 13 .35 A C
ATOM 51 CD1 ILE A 7 20 .794 25 .294 2 .972 1 .00 14 .08 A C
ATOM 52 CG2 ILE A 7 20 .511 24 .072 5 .873 1 .00 12 .51 A C
ATOM 53 C ILE A 7 21 .970 21 .664 6 .305 1 .00 15 .04 A C
ATOM 54 O ILE A 7 22 .273 21 .906 7, .469 1 .00 13 .35 A O
ATOM 55 N VAL A 8 21 .320 20 .558 5, .952 1 .00 15 .03 A N
ATOM 56 CA VAL A 8 20 .795 19 .628 6 .969 1 .00 14 .89 A C
ATOM 57 CB VAL A 8 19 .419 19 .047 6 .545 1 .00 14 .63 A C
ATOM 58 CGI VAL A 8 18 .472 20 .135 6 .246 1 .00 14 .63 A C
ATOM 59 CG2 VAL A 8 19 .526 18 .151 5 .333 1 .00 15 .54 A C
ATOM 60 C VAL A 8 21 .770 18 .511 7, .356 1 .00 14 .75 A C
ATOM 61 O VAL A 8 21 .438 17 .645 8, .168 1 .00 15 .23 A O
ATOM 62 N LYS A 9 22 .983 18 .568 6, .804 1 .00 14 .02 A N
ATOM 63 CA LYS A 9 24 .061 17 .627 7, .118 1, .00 14 .55 A C
ATOM 64 CB LYS A 9 24 .374 17 .560 8, .621 1, .00 15 .28 A C
ATOM 65 CG LYS A 9 24 .553 18 .888 9, .299 1, .00 18, .34 A C
ATOM 66 CD LYS A 9 25 .757 19 .608 8, .810 1, .00 23, .66 A C
ATOM 67 CE LYS A 9 26 .025 20 .904 9. .618 1, .00 28, .33 A C
ATOM 68 NZ LYS A 9 27, .283 21 .559 9. .079 1, .00 31, .91 A N
ATOM 69 C LYS A 9 23, .798 16 .226 6. .616 1. .00 13, .77 A C
ATOM 70 O LYS A 9 24, .391 15, .256 7. .132 1. .00 13, .96 A O
ATOM 71 N ALA A 10 22, .979 16, .109 5. ,569 1. .00 13. .98 A N
ATOM 72 CA ALA A 10 22 .816 14 .830 4. .886 1, .00 14 , .34 A C
ATOM 73 CB ALA A 10 21 .649 14 .866 3. .848 1, .00 14, .47 A C
ATOM 74 C ALA A 10 24, .141 14 .437 4. .205 1, .00 14 , .55 A C
ATOM 75 O ALA A 10 24, .409 13 .264 4. .015 1. .00 13. .73 A O
ATOM 76 N ASP A 11 24, .967 15 .423 3. .860 1. .00 16, .04 A N
ATOM 77 CA ASP A 11 26, .278 15, .153 3. .265 1. .00 17, .11 A C
ATOM 78 CB ASP A 11 26, .899 16, .419 2. ,667 1. .00 17. .53 A C
ATOM 79 CG ASP A 11 27, .059 17, .547 3. ,680 1. .00 19. .89 A C
ATOM 80 OD1 ASP A 11 27, .845 18, .461 3. ,375 1. ,00 23. ,81 A O
ATOM 81 OD2 ASP A 11 26, .434 17, .635 4. ,773 1. ,00 20. .19 A O
ATOM 82 C ASP A 11 27, .219 14, .489 4. ,285 1. ,00 17. .57 A C
ATOM 83 O ASP A 11 27, .941 13, .540 3. ,947 1. .00 17. .08 A O
ATOM 84 N VAL A 12 27. .153 14, .945 5. ,528 1. ,00 17. .31 A N
ATOM 85 CA VAL A 12 27, .926 14, .338 6. ,607 1. ,00 17. .86 A C
ATOM 86 CB VAL A 12 27, .850 15, .193 7. ,893 1. ,00 18. ,12 A C
ATOM 87 CGI VAL A 12 28, .577 14 .533 9. .081 1, .00 18, .00 A C
ATOM 88 CG2 VAL A 12 28, .385 16 .633 7. .631 1, .00 19. .36 A C
ATOM 89 C VAL A 12 27, .428 12, .898 6. .835 1. .00 18, .14 A C
ATOM 90 O VAL A 12 28, .233 11, .956 6. .925 1. .00 18, .38 A O
ATOM 91 N ALA A 13 26, .117 12, .696 6. ,870 1. .00 17. .13 A N
ATOM 92 CA ALA A 13 25, .572 11, .353 7. ,076 1. .00 17, .08 A C
ATOM 93 CB ALA A 13 24. .070 11, .400 7. ,101 1. .00 17, .00 A C
ATOM 94 C ALA A 13 26, .044 10, .394 5. ,981 1. ,00 17 , .57 A C
ATOM 95 O ALA A 13 26, .472 9, .237 6. ,254 1. .00 16. .79 A O
ATOM 96 N GLN A 14 25. .934 10, .862 4. ,740 1. ,00 17. ,24 A N
ATOM 97 CA GLN A 14 26. .420 10, .107 3. ,582 1. ,00 17. .55 A C
ATOM 98 CB GLN A 14 25. ,972 10, .825 2. 309 1. ,00 17. ,74 A C
ATOM 99 CG GLN A 14 24. ,485 10. .673 2. 031 1. ,00 17. ,61 A C
ATOM 100 CD GLN A 14 23. ,995 11. .535 0. 887 1. ,00 20. ,02 A C
ATOM 101 OEl GLN A 14 24. ,788 11. .949 0. 028 1. ,00 19. .60 A O
ATOM 102 NE2 GLN A 14 22. ,679 11. .789 0. 850 1. ,00 19. ,07 A N
ATOM 103 C GLN A 14 27, .949 9, .876 3. ,576 1. .00 18, .95 A C
ATOM 104 O GLN A 14 28, .413 8, .729 3. ,489 1. .00 18, .61 A O ATOM 105 N ASN A 15 28.730 10.950 3..658 1.00 19.73 A N
ATOM 106 CA ASN A 15 30 .185 10 .847 3. .469 1, .00 20 .71 A C
ATOM 107 CB ASN A 15 30 .828 12 .222 3. .244 1, .00 20 .45 A C
ATOM 108 CG ASN A 15 30 .404 12, .869 1. .959 1, .00 22, .21 A C
ATOM 109 OD1 ASN A 15 30 .098 12 .201 0, .976 1 .00 25 .39 A 0
ATOM 110 ND2 ASN A 15 30 .390 14 .182 1, .953 1 .00 23 .97 A N
ATOM 111 C ASN A 15 30 .865 10 .185 4. .653 1 .00 20 .49 A C
ATOM 112 O ASN A 15 31 .705 9 .362 4. .469 1 .00 21 .06 A 0
ATOM 113 N ASN A 16 30 .495 10 .559 5. .869 1, .00 21 .00 A N
ATOM 114 CA ASN A 16 31 .148 10 .056 7, .073 1 .00 21 .90 A C
ATOM 115 CB ASN A 16 31 .205 11 .146 8, .136 1 .00 22 .29 A C
ATOM 116 CG ASN A 16 32 .100 12 .313 7. .751 1, .00 26 .21 A C
ATOM 117 OD1 ASN A 16 32 .261 13 .260 8. .533 1, .00 32 .71 A 0
ATOM 118 ND2 ASN A 16 32, .672 12, .268 6. .567 1, .00 28, .57 A N
ATOM 119 C ASN A 16 30 .491 8 .811 7, .692 1 .00 21 .95 A C
ATOM 120 O ASN A 16 31 .152 8 .065 8, .404 1 .00 22 .21 A 0
ATOM 121 N PHE A 17 29 .203 8 .578 7. .438 1 .00 20 .66 A N
ATOM 122 CA PHE A 17 28 .550 7 .392 8. .003 1, .00 20, .24 A C
ATOM 123 CB PHE A 17 27 .415 7 , .815 8. .938 1, .00 21, .09 A C
ATOM 124 CG PHE A 17 27 .890 8 .591 10, .134 1 .00 19 .81 A C
ATOM 125 CD1 PHE A 17 28 .110 7 .953 11, .348 1 .00 24 .93 A C
ATOM 126 CE1 PHE A 17 28 .556 8 .679 12. .459 1, .00 25 .33 A C
ATOM 127 CZ PHE A 17 28 .779 10 .016 12. .344 1, .00 23, .90 A C
ATOM 128 CE2 PHE A 17 28 .564 10 .651 11. ,155 1, .00 22 , .65 A C
ATOM 129 CD2 PHE A 17 28, .111 9, .936 10. ,052 1, .00 20. .02 A C
ATOM 130 C PHE A 17 28 .061 6 .385 6, .977 1 .00 19 .13 A C
ATOM 131 O PHE A 17 27 .607 5 .336 7. .337 1 .00 20 .18 A 0
ATOM 132 N GLY A 18 28 .205 6 .685 5. .692 1, .00 17 .84 A N
ATOM 133 CA GLY A 18 27, .740 5, .790 4. .640 1, .00 17, .25 A C
ATOM 134 C GLY A 18 26, .220 5, .654 4. ,496 1, .00 16, .27 A C
ATOM 135 O GLY A 18 25 .755 4 .667 3, .948 1, .00 14 .47 A 0
ATOM 136 N LEU A 19 25 .453 6 .651 4. .955 1, .00 15, .24 A N
ATOM 137 CA LEU A 19 23, .980 6 .550 5. .007 1, .00 14 , .35 A C
ATOM 138 CB LEU A 19 23, .456 7, .222 6. .270 1, .00 14 , .71 A C
ATOM 139 CG LEU A 19 24, .013 6, .680 7. .569 1, .00 15, .58 A C
ATOM 140 CD1 LEU A 19 23 .691 7 .633 8, .721 1 .00 16 .09 A C
ATOM 141 CD2 LEU A 19 23 .417 5, .294 7. .793 1, .00 15, .86 A C
ATOM 142 C LEU A 19 23, .305 7, .203 3. .820 1, .00 13, .82 A C
ATOM 143 O LEU A 19 23, .183 8, .427 3. .775 1, .00 13, .96 A 0
ATOM 144 N TYR A 20 22, .874 6, .400 2. .854 1, .00 13, .81 A N
ATOM 145 CA TYR A 20 22 .156 6 .917 1, .714 1 .00 14 .22 A C
ATOM 146 CB TYR A 20 22 .841 6 .499 0, .386 1 .00 14 .36 A C
ATOM 147 CG TYR A 20 24 .254 7 .034 0. .241 1, .00 14, .09 A C
ATOM 148 CD1 TYR A 20 25, .351 6, .353 0. .792 1, .00 16, .48 A C
ATOM 149 CE1 TYR A 20 26, .661 6, .858 0. .663 1, .00 16, .91 A C
ATOM 150 CZ TYR A 20 26 .859 8 .041 -0, .034 1 .00 18 .57 A C
ATOM 151 OH TYR A 20 28 .126 8 .567 -0, .171 1 .00 21 .21 A 0
ATOM 152 CE2 TYR A 20 25 .788 8 .735 -0. .575 1. .00 17 .45 A C
ATOM 153 CD2 TYR A 20 24, .495 8, .217 -0. .461 1, .00 16 .03 A C
ATOM 154 C TYR A 20 20, .715 6, .433 1. .702 1, .00 14 , .55 A C
ATOM 155 O TYR A 20 19, .994 6, .688 0. .723 1, .00 14. .48 A 0
ATOM 156 N GLY A 21 20 .297 5 .710 2, .746 1 .00 14 .18 A N
ATOM 157 CA GLY A 21 18 .947 5 .172 2. .802 1, .00 14 .23 A C
ATOM 158 C GLY A 21 18, .749 3, .775 2. .207 1, .00 14 .56 A C
ATOM 159 O GLY A 21 17, .611 3, .315 2. .054 1, .00 13 .53 A 0
ATOM 160 N GLN A 22 19, .838 3, .084 1. .883 1, .00 14, .57 A N
ATOM 161 CA GLN A 22 19 .722 1 .726 1, .334 1 .00 14 .82 A C
ATOM 162 CB GLN A 22 21 .095 1 .130 0, .978 1, .00 15 .45 A C
ATOM 163 CG GLN A 22 21, .054 -0 .151 0. .150 1, .00 17 .91 A C
ATOM 164 CD GLN A 22 20, .669 -1, .376 0. .976 1, .00 21 .79 A C
ATOM 165 OEl GLN A 22 20, .892 -1, .414 2. .185 1, .00 22 .42 A 0
ATOM 166 NE2 GLN A 22 20 .091 -2 .379 0, .317 1 .00 23 .11 A N
ATOM 167 C GLN A 22 19 .011 0 .831 2, .331 1 .00 14 .04 A C ATOM 168 O GLN A 22 19.341 0.824 3,.516 1.00 14.39 A o
ATOM 169 N GLY A 23 18 .019 0 .110 1, .836 1 .00 14 .26 A N
ATOM 170 CA GLY A 23 17 .236 -0 .859 2, .628 1 .00 14 .81 A C
ATOM 171 C GLY A 23 15 .957 -0 .245 3 .176 1 .00 14 .12 A C
ATOM 172 O GLY A 23 15 .086 -0 .948 3 .718 1 .00 14 .17 A O
ATOM 173 N GLN A 24 15 .836 1 .077 3 .057 1 .00 13 .54 A N
ATOM 174 CA GLN A 24 14 .620 1 .773 3, .500 1 .00 13 .27 A C
ATOM 175 CB GLN A 24 14 .963 3 .090 4, .182 1 .00 12 .64 A C
ATOM 176 CG GLN A 24 15 .806 2 .945 5, .450 1 .00 13 .46 A C
ATOM 177 CD GLN A 24 15 .150 2 .100 6, .505 1 .00 15 .72 A C
ATOM 178 OEl GLN A 24 14 .015 2 .387 6, .921 1 .00 14 .73 A O
ATOM 179 NE2 GLN A 24 15 .839 1 .026 6, .927 1 .00 13 .89 A N
ATOM 180 C GLN A 24 13 .619 2 .022 2, .352 1 .00 13 .19 A C
ATOM 181 O GLN A 24 14 .005 2 .126 1, .184 1 .00 13 .48 A O
ATOM 182 N ILE A 25 12 .324 2 .066 2, .692 1 .00 13 .28 A N
ATOM 183 CA ILE A 25 11 .280 2 .319 1, .720 1 .00 13 .25 A C
ATOM 184 CB ILE A 25 10, .404 1 .077 1, .507 1 .00 13 .64 A C
ATOM 185 CGI ILE A 25 11, .267 -0 .108 1, .030 1, .00 15 .44 A C
ATOM 186 CD1 ILE A 25 10 .508 -1 .518 0, .962 1 .00 14 .73 A C
ATOM 187 CG2 ILE A 25 9 .303 1 .387 0, .503 1 .00 13 .37 A C
ATOM 188 C ILE A 25 10 .447 3 .491 2, .209 1 .00 13 .24 A C
ATOM 189 O ILE A 25 9, .884 3 .430 3 , .285 1, .00 12 .93 A O
ATOM 190 N VAL A 26 10, .438 4 .573 1. .432 1, .00 12 .43 A N
ATOM 191 CA VAL A 26 9, .656 5 .754 1. .737 1, .00 12 .49 A C
ATOM 192 CB VAL A 26 10, .480 7 .034 1. .585 1, .00 12 .90 A C
ATOM 193 CGI VAL A 26 9, .671 8 .231 2. ,059 1, .00 11 .57 A C
ATOM 194 CG2 VAL A 26 11 .796 6 .928 2, .395 1 .00 15 .53 A C
ATOM 195 C VAL A 26 8 .465 5 .823 0, .804 1 .00 12 .34 A C
ATOM 196 O VAL A 26 8 .601 5 .646 -0, .418 1, .00 11 .99 A O
ATOM 197 N ALA A 27 7, .297 6 .044 1. .387 1, .00 12 .40 A N
ATOM 198 CA ALA A 27 6, .080 6 .289 0. .624 1, .00 12 .49 A C
ATOM 199 CB ALA A 27 4, .846 5 .650 1. .284 1, .00 11 .39 A C
ATOM 200 C ALA A 27 5, .892 7, .790 0. ,546 1. .00 12 .17 A C
ATOM 201 O ALA A 27 6, .077 8. .501 1. .526 1. .00 11 .39 A O
ATOM 202 N VAL A 28 5, .540 8 .243 -0. .643 1, .00 11 .79 A N
ATOM 203 CA VAL A 28 5, .168 9 .612 -0. .910 1, .00 11 .63 A C
ATOM 204 CB VAL A 28 6, .054 10 .176 -2. .003 1, .00 11 .56 A C
ATOM 205 CGI VAL A 28 5, .629 11 .625 -2. .440 1, .00 12 .77 A C
ATOM 206 CG2 VAL A 28 7, .514 10 .079 -1. .594 1, .00 11 .95 A C
ATOM 207 C VAL A 28 3, .729 9 .580 -1. .458 1. .00 11 .23 A C
ATOM 208 O VAL A 28 3, .470 8 .936 -2. ,459 1, .00 10 .72 A O
ATOM 209 N ALA A 29 2, .817 10, .294 -0. ,831 1. .00 10 .64 A N
ATOM 210 CA ALA A 29 1 .468 10 .435 -1. .365 1, .00 11 .32 A C
ATOM 211 CB ALA A 29 0, .441 10 .151 -0, .298 1, .00 11 .33 A C
ATOM 212 C ALA A 29 1, .326 11 .842 -1. .909 1. .00 11 .35 A C
ATOM 213 O ALA A 29 1, .404 12 .826 -1. .161 1, .00 11 .19 A O
ATOM 214 N ASP A 30 1, .186 11 .937 -3. .229 1, .00 11 .71 A N
ATOM 215 CA ASP A 30 1, .266 13 .221 -3. .917 1, .00 11 .52 A C
ATOM 216 CB ASP A 30 2 , .718 13 .715 -3. .958 1 , .00 11 .37 A C
ATOM 217 CG ASP A 30 2 .802 15 .221 -3, .852 1, .00 12 .40 A C
ATOM 218 OD1 ASP A 30 3 .385 15 .726 -2, .871 1, .00 12 .15 A O
ATOM 219 OD2 ASP A 30 2 .226 15 .973 -4 , .682 1, .00 14 .39 A O
ATOM 220 C ASP A 30 0, .665 13 .113 -5. .327 1, .00 12 .41 A C
ATOM 221 O ASP A 30 0, .068 12 .086 -5. .671 1, .00 12 .89 A O
ATOM 222 N THR A 31 0, .811 14 .162 -6. .151 1, .00 12 .52 A N
ATOM 223 CA THR A 31 -0, .004 14 .263 -7. .353 1 , .00 11 .62 A C
ATOM 224 CB THR A 31 0. .302 15 .554 -8. ,182 1. .00 11 .72 A C
ATOM 225 OGl THR A 31 1 .709 15 .702 -8, .423 1, .00 11 .44 A o
ATOM 226 CG2 THR A 31 -0 .099 16 .789 -7, .424 1, .00 11 .85 A C
ATOM 227 C THR A 31 0, .126 13 .041 -8. .225 1, .00 12 .59 A C
ATOM 228 O THR A 31 -0, .868 12 .341 -8. .494 1, .00 12 .63 A O
ATOM 229 N GLY A 32 1, .360 12 .810 -8. .665 1, .00 12 .03 A N
ATOM 230 CA GLY A 32 1, .694 11 .788 -9. .617 1, .00 12 .81 A C ATOM 231 C GLY A 32 3,.202 11..763 -9.,729 1..00 13.,22 A C
ATOI 232 O GLY A 32 3. ,885 12. ,607 -9. 135 1. 00 12. 83 A O
ATOM 233 N LEU A 33 3. .711 10. .813 -10. 501 1. 00 13. 41 A N
ATOM 234 CA LEU A 33 5, .139 10. ,622 -10. 678 1. ,00 13. 86 A C
ATOM 235 CB LEU A 33 5, .625 9. .397 -9. ,899 1. ,00 13. ,74 A C
ATOM 236 CG LEU A 33 7, .148 9. .234 -9. ,900 1. ,00 14. .12 A C
ATOM 237 CD1 LEU A 33 7, .768 10. .273 -8. ,964 1. ,00 13. ,99 A C
ATOM 238 CD2 LEU A 33 7. .497 7. .818 -9. ,437 1. ,00 15. .42 A C
ATOM 239 C LEU A 33 5, .517 10. .505 -12. ,151 1. ,00 13. ,89 A C
ATOM 240 O LEU A 33 5, .374 9. .444 -12. ,765 1. ,00 14. ,14 A O
ATOM 241 N ASP A 34 6, .009 11. .612 -12. .696 1. ,00 14. ,55 A N
ATOM 242 CA ASP A 34 6, .455 11, .701 -14. ,087 1. .00 14. .62 A C
ATOM 243 CB ASP A 34 7, .899 11, .201 -14. ,224 1. .00 14. .72 A C
ATOM 244 CG ASP A 34 8, .516 11, .532 -15. .598 1. .00 15, .30 A C
ATOM 245 OD1 ASP A 34 9, .260 10. .694 -16. .148 1. ,00 14. ,31 A O
ATOM 246 OD2 ASP A 34 8, .268 12, .602 -16. ,207 1. .00 17. .33 A O
ATOM 247 C ASP A 34 5, .470 11, .016 -15. .060 1. .00 14. .94 A C
ATOM 248 O ASP A 34 4, .297 11. .415 -15. .124 1. .00 15. .39 A O
ATOM 249 N THR A 35 5, .927 10. .013 -15. .816 1, .00 16. .25 A N
ATOM 250 CA THR A 35 5 .083 9, .340 -16, .813 1, .00 16, .50 A C
ATOM 251 CB THR A 35 5. .912 8, .471 -17, .786 1, .00 17, .03 A C
ATOM 252 OGl THR A 35 6, .700 7 , .514 -17. ,051 1. .00 17. .34 A O
ATOM 253 CG2 THR A 35 6, .922 9, .300 -18. .593 1. .00 17. .53 A C
ATOM 254 C THR A 35 4, .005 8, .437 -16. .229 1. .00 17. .26 A C
ATOM 255 O THR A 35 3, .111 7, .992 -16. .946 1, .00 15, .49 A O
ATOM 256 N GLY A 36 4 .104 8, .104 -14 , .948 1, .00 16, .59 A N
ATOM 257 CA GLY A 36 3 .094 7, .259 -14, .360 1, .00 16, .76 A C
ATOM 258 C GLY A 36 3, .308 5, .802 -14. .660 1. .00 17. .58 A C
ATOM 259 O GLY A 36 2, .432 4 , .984 -14. .383 1. .00 17 , .55 A O
ATOM 260 N ARG A 37 4, .473 5, .465 -15. .200 1. .00 18, .31 A N
ATOM 261 CA ARG A 37 4 .748 4 , .091 -15, .575 1, .00 19, .42 A C
ATOM 262 CB ARG A 37 4 .763 3 .940 -17, .088 1, .00 20, .37 A C
ATOM 263 CG ARG A 37 3 .436 4 .298 -17, .742 1, .00 23 .71 A C
ATOM 264 CD ARG A 37 3 .283 3 .740 -19, .140 1, .00 31, .29 A C
ATOM 265 NE ARG A 37 4, .324 4. .233 -20. .024 1. .00 34, .29 A N
ATOM 266 CZ ARG A 37 4, .322 5, .434 -20, .575 1, .00 38, .63 A C
ATOM 267 NH1 ARG A 37 5, .331 5, .792 -21. .361 1, .00 39, .90 A N
ATOM 268 NH2 ARG A 37 3, .305 6 .273 -20, .362 1, .00 40 .23 A N
ATOM 269 C ARG A 37 6 .072 3 .661 -14, .998 1 .00 19 .41 A C
ATOM 270 O ARG A 37 7 .065 4 .354 -15, .150 1 .00 18 .12 A O
ATOM 271 N ASN A 38 6 .067 2 .506 -14 .334 1 .00 19 .47 A N
ATOM 272 CA ASN A 38 7 .254 1. .998 -13, .703 1, .00 20 .37 A C
ATOM 273 CB ASN A 38 6 .917 1. .215 -12, .431 1 .00 20 .71 A C
ATOM 274 CG ASN A 38 8, .161 0 .841 -11, .658 1 .00 21 .12 A C
ATOM 275 OD1 ASN A 38 9 .248 1 .337 -11, .968 1 .00 18 .41 A O
ATOM 276 ND2 ASN A 38 8 .023 -0 .072 -10 .684 1 .00 20 .60 A N
ATOM 277 C ASN A 38 7 .984 1 .134 -14 .700 1 .00 21 .21 A C
ATOM 278 O ASN A 38 7, .918 -0. .099 -14, .638 1 .00 21 .03 A O
ATOM 279 N ASP A 39 8 .659 1 .806 -15, .625 1 .00 21 .69 A N
ATOM 280 CA ASP A 39 9 .363 1 .158 -16, .718 1 .00 23 .22 A C
ATOM 281 CB ASP A 39 8. .405 0. .839 -17 .882 1 .00 23 .19 A C
ATOM 282 CG ASP A 39 7 .806 2 .082 -18 .526 1 .00 24 .66 A C
ATOM 283 OD1 ASP A 39 6 .796 1 .945 -19 .248 1 .00 26 .50 A O
ATOM 284 OD2 ASP A 39 8 .246 3 .239 -18 .372 1 .00 27 .03 A O
ATOM 285 C ASP A 39 10 .480 2 .075 -17, .156 1 .00 24 .00 A C
ATOM 286 O ASP A 39 10 .843 3 .004 -16, .434 1 .00 23 .68 A O
ATOM 287 N SER A 40 11 .003 1 .832 -18 .355 1 .00 24 .67 A N
ATOM 288 CA SER A 40 12 .166 2 .539 -18 .847 1 .00 24 .80 A C
ATOM 289 CB SER A 40 12 .777 1 .766 -20 .041 1 .00 25 .30 A C
ATOM 290 OG SER A 40 11 .925 1 .881 -21 .163 1 .00 25 .60 A O
ATOM 291 C SER A 40 11 .815 3 .984 -19 .228 1 .00 23 .51 A C
ATOM 292 O SER A 40 12 .687 4 .805 -19 .375 1 .00 24 .41 A O
ATOM 293 N SER A 41 10 .532 4 .308 -19 .317 1 .00 23 .14 A N ATOM 294 CA SER A 41 10,.097 5..670 -19,,621 1,.00 21.75 A C
ATOM 295 CB SER A 41 8, .620 5 .679 -20, .037 1, .00 22 .84 A C
ATOM 296 OG SER A 41 7, .725 5 .739 -18, .919 1 .00 21 .43 A O
ATOM 297 C SER A 41 10. .262 6, .639 -18. .427 1. .00 21. .13 A C
ATOM 298 O SER A 41 10. .299 7 .863 -18. .603 1, ,00 19, ,88 A O
ATOM 299 N MET A 42 10. .359 6, .079 -17. .223 1, ,00 19, .68 A N
ATOM 300 CA MET A 42 10. .381 6 .882 -15, ,996 1, .00 18. .70 A C
ATOM 301 CB MET A 42 10. .295 5, .949 -14, .782 1, .00 18, .20 A C
ATOM 302 CG MET A 42 10. .451 6 .626 -13, .423 1, .00 17, .87 A C
ATOM 303 SD MET A 42 9. .190 7 .804 -13, .030 1, .00 16, .31 A S
ATOM 304 CE MET A 42 7. .658 6 .844 -13, .134 1, .00 15, .38 A C
ATOM 305 C MET A 42 11. .607 7 .779 -15, .897 1, .00 17, .89 A C
ATOM 306 O MET A 42 12. .728 7 .390 -16, .223 1, .00 17, .28 A O
ATOM 307 N HIS A 43 11. .381 8. .998 -15. .421 1, .00 17, .69 A N
ATOM 308 CA HIS A 43 12, .479 9 .903 -15, .081 1, .00 17, .38 A C
ATOM 309 CB HIS A 43 11. .942 11 .020 -14, .196 1, .00 17 .29 A C
ATOM 310 CG HIS A 43 12, .896 12 .155 -13, .981 1, .00 16 .73 A C
ATOM 311 NDl HIS A 43 12, .576 13 .456 -14, .321 1 .00 16 .98 A N
ATOM 312 CE1 HIS A 43 13. .566 14 .257 -13, .971 1 .00 13 .61 A C
ATOM 313 NE2 HIS A 43 14, .521 13 .523 -13, .426 1 .00 17 .49 A N
ATOM 314 CD2 HIS A 43 14, .113 12 .207 -13 .397 1 .00 13 .70 A C
ATOM 315 C HIS A 43 13, .647 9 .209 -14 .381 1 .00 16 .64 A C
ATOM 316 O HIS A 43 13, .453 8 .389 -13 .479 1 .00 15 .82 A O
ATOM 317 N GLU A 44 14, .858 9 .559 -14 .818 1 .00 16 .35 A N
ATOM 318 CA GLU A 44 16 .112 8 .985 -14, .358 1 .00 16 .74 A C
ATOM 319 CB GLU A 44 17 .293 9 .763 -14 .988 1 .00 17 .71 A C
ATOM 320 CG GLU A 44 17, .268 11 .270 -14, .753 1, .00 18 .20 A C
ATOM 321 CD GLU A 44 18, .445 12 .004 -15, .418 1, .00 22 .20 A C
ATOM 322 OEl GLU A 44 18, .997 11 .455 -16, .397 1, .00 20 .94 A O
ATOM 323 OE2 GLU A 44 18, .843 13 .110 -14, .933 1, .00 20 .93 A O
ATOM 324 C GLU A 44 16, .280 8 .982 -12, .823 1, .00 17 .11 A C
ATOM 325 O GLU A 44 16. .944 8. .104 -12, .259 1 .00 17 .08 A O
ATOM 326 N ALA A 45 15, .665 9 .954 -12, .152 1, .00 16 .39 A N
ATOM 327 CA ALA A 45 15, .774 10 .061 -10, .696 1, .00 16 .01 A C
ATOM 328 CB ALA A 45 15, .122 11 .354 -10, .198 1 .00 15 .44 A C
ATOM 329 C ALA A 45 15, .155 8 .864 -9, .971 1 .00 16 .39 A C
ATOM 330 O ALA A 45 15, .538 8 .564 -8, .857 1 .00 14 .42 A O
ATOM 331 N PHE A 46 14, .184 8 .218 -10 .595 1 .00 16 .01 A N
ATOM 332 CA PHE A 46 13, .411 7 .139 -9 .971 1 .00 16 .28 A C
ATOM 333 CB PHE A 46 11, .958 7 .562 -9 .882 1 .00 16 .14 A C
ATOM 334 CG PHE A 46 11, .780 8 .959 -9 .396 1 .00 14 .90 A C
ATOM 335 CD1 PHE A 46 12 .036 9 .275 -8 .078 1. .00 14 .17 A C
ATOM 336 CE1 PHE A 46 11 .897 10 .586 -7 .628 1 .00 13 .19 A C
ATOM 337 CZ PHE A 46 11 .525 11 .592 -8 .504 1 .00 14 .92 A C
ATOM 338 CE2 PHE A 46 11 .291 11 .299 -9 .809 1 .00 16 .01 A C
ATOM 339 CD2 PHE A 46 11 .416 9 .971 -10 .261 1 .00 15 .93 A C
ATOM 340 C PHE A 46 13 .466 5 .791 -10 .697 1 .00 17 .23 A C
ATOM 341 O PHE A 46 13 .017 4 .764 -10 .172 1 .00 16 .06 A O
ATOM 342 N ARG A 47 13 .986 5 .781 -11 .917 1 .00 18 .87 A N
ATOM 343 CA ARG A 47 13, .963 4 .566 -12, .723 1, .00 20 .32 A C
ATOM 344 CB ARG A 47 14, .659 4 .833 -14, .062 1, .00 21 .00 A C
ATOM 345 CG ARG A 47 14, .309 3. .871 -15. , 173 1, .00 24 .13 A C
ATOM 346 CD ARG A 47 14, .468 4 .517 -16, .570 1, .00 28 .53 A C
ATOM 347 NE ARG A 47 15, .803 5 .031 -16, ,813 1 .00 32 .22 A N
ATOM 348 CZ ARG A 47 16, .105 6 .229 -17, ,359 1 .00 34 .45 A C
ATOM 349 NH1 ARG A 47 15, ,171 7 .109 -17, .703 1, .00 33 .97 A N
ATOM 350 NH2 ARG A 47 17, .384 6 .558 -17, .527 1 .00 33 .82 A N
ATOM 351 C ARG A 47 14, .674 3 .437 -12, .000 1 .00 20 .49 A C
ATOM 352 O ARG A 47 15, .784 3 .619 -11, .523 1 .00 21 .38 A O
ATOM 353 N GLY A 48 14 .032 2 .280 -11 .898 1 .00 21 .45 A N
ATOM 354 CA GLY A 48 14 .642 1 .105 -11 .274 1 .00 21 .59 A C
ATOM 355 C GLY A 48 14 .583 1 .091 -9 .741 1 .00 21 .98 A C
ATOM 356 O GLY A 48 15 .072 0 .145 -9 .102 1 .00 21 .74 A O ATOM 357 N LYS A 49 13.984 2.117 -9,.136 1.00 21.05 A N
ATOM 358 CA LYS A 49 13 .950 2 .197 -7, ,662 1 .00 20 .90 A C
ATOM 359 CB LYS A 49 14 .915 3 .305 -7, .180 1 .00 22 .03 A C
ATOM 360 CG LYS A 49 1 .366 4 .713 -7, .161 1 .00 24 .83 A C
ATOM 361 CD LYS A 49 15 .447 5 .815 -6, .761 1 .00 27 .45 A C
ATOM 362 CE LYS A 49 15 .957 5 .680 -5, .358 1 .00 27 .82 A C
ATOM 363 NZ LYS A 49 17 .024 4 .667 -5, .220 1 .00 28 .25 A N
ATOM 364 C LYS A 49 12 .523 2 .329 -7, .077 1 .00 19 .90 A C
ATOM 365 O LYS A 49 12 .339 2 .667 -5, .890 1 .00 19 .91 A O
ATOM 366 N ILE A 50 11 .523 1 .999 -7 .900 1 .00 18 .63 A N
ATOM 367 CA ILE A 50 10 .121 2 .078 -7, .533 1 .00 17 .48 A C
ATOM 368 CB ILE A 50 9 .284 2 .650 -8 .695 1 .00 17 .67 A C
ATOM 369 CGI ILE A 50 9 .738 4 .076 -9 .050 1 .00 17 .24 A C
ATOM 370 CD1 ILE A 50 9 .083 4 .630 -10 .302 1 .00 17 .34 A C
ATOM 371 CG2 ILE A 50 7, .807 2 .723 -8, ,319 1, .00 17 .29 A C
ATOM 372 C ILE A 50 9 .562 0 .730 -7, .090 1, .00 17 .96 A C
ATOM 373 O ILE A 50 9 .339 -0 .161 -7, .909 1, .00 18 .69 A O
ATOM 374 N THR A 51 9 .355 0 .583 -5, .784 1, .00 17 .09 A N
ATOM 375 CA THR A 51 8, .731 -0 .601 -5, ,218 1, .00 17 .60 A C
ATOM 376 CB THR A 51 8 .700 -0 .423 -3, .690 1, .00 18 .38 A C
ATOM 377 OGl THR A 51 10 .033 -0 .380 -3. .205 1, .00 17 .39 A O
ATOM 378 CG2 THR A 51 8 .054 -1 .617 -3, .014 1, .00 17 .34 A C
ATOM 379 C THR A 51 7, .301 -0 .746 -5, .646 1, .00 17 .50 A C
ATOM 380 O THR A 51 6 .827 -1 .834 -5. .903 1, .00 18 .10 A O
ATOM 381 N ALA A 52 6, .578 0 .369 -5, ,670 1, .00 16 .93 A N
ATOM 382 CA ALA A 52 5, .179 0 .338 -6, .052 1, .00 17 .34 A C
ATOM 383 CB ALA A 52 4 .314 -0 .132 -4, .884 1, .00 17 .41 A C
ATOM 384 C ALA A 52 4, .753 1 .725 -6, ,501 1, .00 17 .46 A C
ATOM 385 O ALA A 52 5 .187 2 .730 -5. .928 1 .00 16 .50 A O
ATOM 386 N LEU A 53 3 .921 1 .760 -7, .539 1, .00 17 .19 A N
ATOM 387 CA LEU A 53 3 .369 2 .987 -8, .081 1, .00 16 .89 A C
ATOM 388 CB LEU A 53 4 .004 3 .309 -9, .430 1 .00 16 .66 A C
ATOM 389 CG LEU A 53 3 .490 4 .525 -10. .224 1 .00 16 .83 A C
ATOM 390 CD1 LEU A 53 3 .523 5 .796 -9, .401 1, .00 15 .83 A C
ATOM 391 CD2 LEU A 53 4 .303 4 .720 -11, ,476 1 .00 17 .71 A C
ATOM 392 C LEU A 53 1 .868 2 .779 -8, .212 1 .00 17 .28 A C
ATOM 393 O LEU A 53 1 .421 2 .057 -9, .097 1 .00 17 .43 A O
ATOM 394 N TYR A 54 1 .101 3 .393 -7, ,303 1 .00 17 .28 A N
ATOM 395 CA TYR A 54 -0 .350 3 .200 -7, .230 1 .00 16 .87 A C
ATOM 396 CB TYR A 54 -0 .774 2 .944 -5, .789 1 .00 16 .88 A C
ATOM 397 CG TYR A 54 -0 .268 1 .679 -5, .144 1 .00 15 .63 A C
ATOM 398 CD1 TYR A 54 -0 .411 0 .448 -5. .770 1 .00 15 .86 A C
ATOM 399 CE1 TYR A 54 0 .037 -0 .698 -5, .192 1 .00 15 .12 A C
ATOM 400 CZ TYR A 54 0 .666 -0 .647 -3, .946 1 .00 15 .32 A C
ATOM 401 OH TYR A 54 1 .093 -1 .815 -3, .374 1 .00 14 .97 A O
ATOM 402 CE2 TYR A 54 0 .856 0 .558 -3, .312 1 .00 15 .70 A C
ATOM 403 CD2 TYR A 54 0 .384 1 .718 -3, .908 1 .00 15 .59 A C
ATOM 404 C TYR A 54 -1 .098 4 .411 -7, .712 1 .00 17 .11 A C
ATOM 405 O TYR A 54 -0 .733 5 .546 -7, .387 1 .00 16 .78 A O
ATOM 406 N ALA A 55 -2 .161 4 .184 -8, .483 1 .00 17 .01 A N
ATOM 407 CA ALA A 55 -3 .032 5 .260 -8. .926 1 .00 17 .49 A C
ATOM 408 CB ALA A 55 -3 .355 5 .094 -10, .437 1 .00 17 .43 A C
ATOM 409 C ALA A 55 -4 .323 5 .272 -8, .100 1 .00 18 .26 A C
ATOM 410 O ALA A 55 -5 .174 4 .400 -8. .269 1 .00 19 .60 A O
ATOM 411 N LEU A 56 -4 .481 6 .267 -7, .230 1 .00 17 .69 A N
ATOM 412 CA LEU A 56 -5 .641 6 .353 -6, ,368 1 .00 17 .32 A C
ATOM 413 CB LEU A 56 -5 .224 6 .779 -4, .965 1 .00 16 .99 A C
ATOM 414 CG LEU A 56 -4 .452 5 .752 -4, .129 1 .00 17 .97 A C
ATOM 415 CD1 LEU A 56 -3 .120 5 .532 -4. .719 1 .00 20 .33 A C
ATOM 416 CD2 LEU A 56 -4 .329 6 .225 -2. .662 1 .00 19 .18 A C
ATOM 417 C LEU A 56 -6 .662 7 .360 -6, .867 1 .00 17 .28 A C
ATOM 418 O LEU A 56 -7 .839 7 .192 -6. ,653 1, .00 17 .95 A O
ATOM 419 N GLY A 57 -6 .204 8 .430 -7, .485 1, .00 17 .26 A N ATOM 420 CA GLY A 57 -7..068 9,,541 -7.,802 1.,00 17 ,.53 A C
ATOM 421 C GLY A 57 -7. .662 9, .430 -9. ,199 1. ,00 17, .74 A c
ATOM 422 O GLY A 57 -8. ,758 9. .905 -9. ,446 1. ,00 17, .69 A O
ATOM 423 N ARG A 58 -6. ,921 8. .825 -10. 109 1. 00 18. ,54 A N
ATOM 424 CA ARG A 58 -7. ,361 8. .659 -11. 502 1. ,00 19. ,44 A C
ATOM 425 CB ARG A 58 -6. ,572 9. .555 -12. ,466 1. ,00 18. .56 A C
ATOM 426 CG ARG A 58 -6. ,873 11. .036 -12. ,371 1. ,00 18. .16 A C
ATOM 427 CD ARG A 58 -5. ,685 11, .912 -12. ,787 1. ,00 17. .99 A C
ATOM 428 NE ARG A 58 -4. ,505 11. .593 -11. ,990 1. ,00 17. .16 A N
ATOM 429 CZ ARG A 58 -3. ,248 11, .716 -12. ,392 1. ,00 18. .78 A C
ATOM 430 NH1 ARG A 58 -2. .967 12 , .194 -13. ,591 1. ,00 18. .96 A N
ATOM 431 NH2 ARG A 58 -2. .253 11, ,339 -11. ,584 1. ,00 17, .60 A N
ATOM 432 C ARG A 58 -7, ,123 7, .240 -11. ,909 1. ,00 19, .97 A C
ATOM 433 O ARG A 58 -6, .007 6. .754 -11. .878 1. ,00 20, .06 A O
ATOM 434 N THR A 59 -8, ,183 6, .575 -12. ,324 1. ,00 22 , .06 A N
ATOM 435 CA THR A 59 -8, .091 5, .180 -12. .688 1. .00 22 , .88 A C
ATOM 436 CB THR A 59 -9, .479 4 , .693 -13. .142 1. .00 24, .04 A C
ATOM 437 OGl THR A 59 -10, .330 4 .643 -11. .984 1. .00 25, .24 A O
ATOM 438 CG2 THR A 59 -9, .406 3 .250 -13. .657 1. .00 25, .24 A C
ATOM 439 C THR A 59 -7, .009 4 .919 -13, .733 1. .00 22, .06 A C
ATOM 440 O THR A 59 -7, .020 5 .482 -14, .835 1, ,00 22 .93 A O
ATOM 441 N ASN A 60 -6, .074 4 .068 -13. ,332 1, .00 21 .27 A N
ATOM 442 CA ASN A 60 -4, .939 3 .618 -14, ,124 1, .00 21 .57 A C
ATOM 443 CB ASN A 60 -5, .400 2 .788 -15, ,326 1, .00 22 .51 A C
ATOM 444 CG ASN A 60 -5, .861 1 .401 -14, .927 1, .00 24 .76 A C
ATOM 445 OD1 ASN A 60 -5. .546 0 .908 -13, .835 1, .00 27 .82 A 0
ATOM 446 ND2 ASN A 60 -6. .624 0, .773 -15. .801 1. .00 25. .97 A N
ATOM 447 C ASN A 60 -4. .038 4 , .744 -14. .614 1. .00 20, .35 A C
ATOM 448 O ASN A 60 -3. .369 4 .589 -15. .629 1. .00 20, .71 A 0
ATOM 449 N ASN A 61 -4, ,023 5, .852 -13. ,897 1. .00 18, .43 A N
ATOM 450 CA ASN A 61 -3, .217 6 .996 -14. .300 1. .00 18, .53 A C
ATOM 451 CB ASN A 61 -4. .095 8 .062 -14, .972 1. .00 17, .54 A C
ATOM 452 CG ASN A 61 -3, .278 9 .194 -15, .580 1, ,00 19 .62 A C
ATOM 453 OD1 ASN A 61 -3. .832 10 .171 -16, .141 1, ,00 22 .44 A O
ATOM 454 ND2 ASN A 61 -1, .968 9 .081 -15, .481 1. .00 15 .52 A N
ATOM 455 C ASN A 61 -2, ,520 7 .586 -13, ,088 1, .00 16 .75 A C
ATOM 456 O ASN A 61 -3, .159 8 .213 -12, .260 1, .00 16 .00 A O
ATOM 457 N ALA A 62 -1. .219 7 .357 -12, .988 1, .00 16 .29 A N
ATOM 458 CA ALA A 62 -0, .418 7 .910 -11, .902 1, .00 16 .34 A C
ATOM 459 CB ALA A 62 0 .310 6 .804 -11, .183 1, .00 16 .55 A C
ATOM 460 C ALA A 62 0 .584 8 .948 -12, .405 1, .00 16 .52 A C
ATOM 461 O ALA A 62 1 .583 9 .221 -11, .728 1, .00 15 .61 A O
ATOM 462 N ASN A 63 0 .344 9 .515 -13 .593 1 .00 15 .91 A N
ATOM 463 CA ASN A 63 1 .276 10 .465 -14 .157 1 .00 15 .75 A C
ATOM 464 CB ASN A 63 1 .251 10 .471 -15 .720 1 .00 15 .36 A C
ATOM 465 CG ASN A 63 0 .043 11 .165 -16 .307 1 .00 16 .00 A C
ATOM 466 OD1 ASN A 63 -0 .617 11 .982 -15 .643 1 .00 14 .50 A O
ATOM 467 ND2 ASN A 63 -0 .274 10 .833 -17 .584 1 .00 15 .36 A N
ATOM 468 C ASN A 63 1. .115 11 .858 -13 .518 1 .00 15 .21 A C
ATOM 469 O ASN A 63 0, .168 12 .108 -12, .762 1, .00 15 .63 A O
ATOM 470 N ASP A 64 2, .047 12 .753 -13, .828 1 .00 15 .12 A N
ATOM 471 CA ASP A 64 2 .192 14 .015 -13, .102 1 .00 15 .29 A C
ATOM 472 CB ASP A 64 3 .450 13 .990 -12, .233 1 .00 14 .59 A C
ATOM 473 CG ASP A 64 3 .532 15 .161 -11, .300 1 .00 15 .31 A C
ATOM 474 OD1 ASP A 64 2 .476 15 .813 -11, .058 1 .00 14 .15 A O
ATOM 475 OD2 ASP A 64 4 .626 15 .516 -10 .776 1 .00 14 .37 A O
ATOM 476 C ASP A 64 2 .236 15 .206 -14 .061 1 .00 15 .53 A C
ATOM 477 O ASP A 64 3 .315 15 .713 -14 .423 1 .00 16 .54 A O
ATOM 478 N PRO A 65 1 .065 15 .644 -14 .476 1 .00 16 .18 A N
ATOM 479 CA PRO A 65 0 .950 16 .813 -15 .343 1 .00 17 .33 A C
ATOM 480 CB PRO A 65 -0 .509 16 .776 -15 .807 1 .00 17 .19 A C
ATOM 481 CG PRO A 65 -1 .225 15 .953 -14 .808 1 .00 17 .73 A C
ATOM 482 CD PRO A 65 -0 .249 15 .043 -14 .172 1 .00 17 .21 A C ATOM 483 C PRO A 65 1.228 18.102 -14.607 1.00 17.72 A C
ATOM 484 O PRO A 65 1 .515 19 .081 -15 .250 1 .00 17 .98 A O
ATOM 485 N ASN A 66 1 .150 18 .065 -13 .279 1 .00 18 .27 A N
ATOM 486 CA ASN A 66 1 .314 19 .217 -12 .426 1 .00 19 .24 A C
ATOM 487 CB ASN A 66 0 .536 18 .958 -11 .111 1 .00 20 .48 A C
ATOM 488 CG ASN A 66 0 .790 19 .993 -10 .068 1 .00 22 .89 A C
ATOM 489 OD1 ASN A 66 1 .942 20 .281 -9 .721 1 .00 23 .54 A O
ATOM 490 ND2 ASN A 66 -0 .287 20 .591 -9 .566 1 .00 25 .20 A N
ATOM 491 C ASN A 66 2 .806 19 .457 -12 .153 1 .00 18 .71 A C
ATOM 492 O ASN A 66 3 .314 20 .549 -12 .353 1 .00 18 .84 A O
ATOM 493 N GLY A 67 3 .500 18 .426 -11 .698 1 .00 17 .55 A N
ATOM 494 CA GLY A 67 4 .917 18 .503 -11 .406 1 .00 16 .38 A C
ATOM 495 C GLY A 67 5 .234 18 .455 -9 .916 1 .00 15 .32 A C
ATOM 496 O GLY A 67 6 .383 18 .167 -9 .542 1 .00 15 .11 A O
ATOM 497 N HIS A 68 4 .230 18 .722 -9 .075 1 .00 13 .44 A N
ATOM 498 CA HIS A 68 4 .406 18 .776 -7 .608 1 .00 12 .51 A C
ATOM 499 CB BHIS A 68 3 .109 19 .121 -6 .891 0 .50 12 .22 A C
ATOM 500 CB AHIS A 68 3 .048 19 .078 -6 .930 0 .50 12 .48 A C
ATOM 501 CG BHIS A 68 3 .266 19 .371 -5 .417 0 .50 10 .61 A C
ATOM 502 CG AHIS A 68 3 .140 19 .398 -5, .464 0 .50 10 .86 A C
ATOM 503 ND1BHIS A 68 2 .741 18 .522 -4, .453 0 .50 5 .34 A N
ATOM 504 NDlAHIS A 68 3 .742 18 .559 -4, .548 0 .50 7 .56 A N
ATOM 505 CE1BHIS A 68 3 .009 19 .016 -3, .254 0 .50 6 .59 A C
ATOM 506 CE1AHIS A 68 3 .674 19 .102 -3, ,341 0 .50 2 .00 A C
ATOM 507 NE2BHIS A 68 3 .678 20 .158 -3, .403 0 .50 7 .98 A N
ATOM 508 NE2AHIS A 68 3 .061 20 .277 -3. .442 0 .50 6 .21 A N
ATOM 509 CD2BHIS A 68 3 .845 20, .405 -4. .745 0 .50 5 .10 A C
ATOM 510 CD2AHIS A 68 2 .697 20, .471 -4. ,756 0 .50 8 .79 A C
ATOM 511 C HIS A 68 4 , .986 17. .474 -7. ,064 1, .00 12 .70 A C
ATOM 512 O HIS A 68 6, .025 17, ,471 -6. ,401 1. .00 12 .91 A O
ATOM 513 N GLY A 69 4 .315 16 .374 -7. .317 1 .00 13 .22 A N
ATOM 514 CA GLY A 69 4 .709 15, .094 -6. .739 1 .00 13 .52 A C
ATOM 515 C GLY A 69 6 .039 14, .574 -7. .181 1, .00 13 .01 A C
ATOM 516 O GLY A 69 6 .751 13, .894 -6. ,418 1, .00 13 .80 A O
ATOM 517 N THR A 70 6, ,391 14, .865 -8. ,432 1, .00 13 .25 A N
ATOM 518 CA THR A 70 7, .651 14, ,425 -8. ,970 1, .00 12 .89 A C
ATOM 519 CB THR A 70 7, .688 14. .638 -10. ,507 1, .00 13. .93 A C
ATOM 520 OGl THR A 70 6, .592 13. .940 -11. ,116 1. .00 14, .34 A O
ATOM 521 CG2 THR A 70 8, .895 13. .977 -11. 110 1. .00 13, .50 A C
ATOM 522 C THR A 70 8. .769 15. .192 -8. 309 1. .00 12, .86 A C
ATOM 523 O THR A 70 9. .816 14. ,622 -8. 013 1. .00 13 , .63 A O
ATOM 524 N HIS A 71 8. .560 16. ,498 -8. 093 1. ,00 12 , ,19 A N
ATOM 525 CA HIS A 71 9. .580 17. ,341 -7. 486 1. ,00 11. .80 A C
ATOM 526 CB HIS A 71 9. ,125 18. ,796 -7. 555 1. ,00 11. .41 A C
ATOM 527 CG BHIS A 71 10. .185 19. ,784 -7. 212 0. ,50 11. .89 A C
ATOM 528 CG AHIS A 71 10. .189 19, ,775 -7. ,181 0. .50 9, .73 A C
ATOM 529 ND1BHIS A 71 10, .926 19, .709 -6. ,050 0. .50 12. .60 A N
ATOM 530 ND1AHIS A 71 10, ,236 20, .388 -5. ,942 0. .50 5, ,89 A N
ATOM 531 CE1BHIS A 71 11, ,791 20, .706 -6. ,025 0. .50 13 , .16 A C
ATOM 532 CE1AHIS A 71 11, .281 21. .192 -5. 898 0. ,50 7. .95 A C
ATOM 533 NE2BHIS A 71 11, .618 21. ,438 -7. 114 0. ,50 14. .18 A N
ATOM 534 NE2AHIS A 71 11, .923 21. ,107 -7. 054 0. .50 10. .14 A N
ATOM 535 CD2BHIS A 71 10. .617 20. .883 -7. 869 0. ,50 10. ,25 A C
ATOM 536 CD2AHIS A 71 11. .258 20. ,231 -7. 874 0. ,50 6. .22 A C
ATOM 537 C HIS A 71 9. .806 16. ,875 -6. 018 1. 00 12. ,35 A C
ATOM 538 O HIS A 71 10. .935 16. ,698 -5. 538 1. 00 12. ,45 A O
ATOM 539 N VAL A 72 8. ,697 16. ,657 -5. 331 1. 00 12. ,33 A N
ATOM 540 CA VAL A 72 8. ,704 16. ,204 -3. 960 1. 00 12. ,62 A C
ATOM 541 CB VAL A 72 7. ,279 16. ,056 -3. 469 1. 00 12. ,75 A C
ATOM 542 CGI VAL A 72 7. ,248 15. 256 -2. 202 1. 00 12. 50 A C
ATOM 543 CG2 VAL A 72 6. ,647 17. 430 -3. 262 1. 00 12. 97 A C
ATOM 544 C VAL A 72 9, ,431 14. .864 -3. 799 1. ,00 13. ,01 A C
ATOM 545 O VAL A 72 10. .333 14. ,707 -2. 947 1. ,00 12. ,02 A 0 ATOM 546 N ALA A 73 9..054 13.888 -4.615 1..00 12.23 A N
ATOM 547 CA ALA A 73 9, .664 12 .572 -4 .521 1 .00 12 .29 A C
ATOM 548 CB ALA A 73 8, .986 11 .617 -5 .440 1 .00 12 .32 A C
ATOM 549 C ALA A 73 11. ,180 12 .682 -4 .850 1, .00 11 .78 A C
ATOM 550 O ALA A 73 11. .985 11 .992 -4 .280 1, .00 11 .60 A O
ATOM 551 N GLY A 74 11. ,553 13 .583 -5 .742 1 .00 12 .04 A N
ATOM 552 CA GLY A 74 12. ,961 13 .760 -6, .069 1, .00 11 .92 A C
ATOM 553 C GLY A 74 13. .768 14 .190 -4 .845 1, .00 12 .13 A C
ATOM 554 O GLY A 74 14. .936 13 .816 -4 .693 1 .00 11 .64 A O
ATOM 555 N SER A 75 13. .157 15 .015 -3 .994 1 .00 12 .08 A N
ATOM 556 CA SER A 75 13, .844 15 .546 -2 .827 1 .00 12 .08 A C
ATOM 557 CB SER A 75 13. .095 16 .748 -2 .267 1, .00 11 .53 A C
ATOM 558 OG SER A 75 13. .254 17 .915 -3 .077 1 .00 13 .29 A O
ATOM 559 C SER A 75 14, .033 14 .477 -1 .739 1 .00 12 .05 A C
ATOM 560 O SER A 75 14, .984 14 .540 -0 .927 1 .00 12 .73 A O
ATOM 561 N VAL A 76 13, .112 13 .524 -1 .676 1 .00 11 .61 A N
ATOM 562 CA VAL A 76 13, .272 12 .407 -0 .748 1 .00 11 .87 A C
ATOM 563 CB VAL A 76 12, .023 11 .519 -0 .691 1 .00 12 .06 A C
ATOM 564 CGI VAL A 76 12, .224 10 .396 0 .324 1 .00 12 .75 A C
ATOM 565 CG2 VAL A 76 10, ,799 12 .319 -0 .316 1, .00 11 .56 A C
ATOM 566 C VAL A 76 14. .415 11 .501 -1 .173 1, .00 12 .11 A C
ATOM 567 O VAL A 76 15, .280 11 .158 -0 .372 1, .00 10 .37 A O
ATOM 568 N LEU A 77 14, ,410 11 .085 -2 .437 1, .00 12 .79 A N
ATOM 569 CA LEU A 77 15. .234 9 .934 -2 .809 1, .00 12 .61 A C
ATOM 570 CB LEU A 77 14, .532 8 .627 -2 .425 1, .00 13 .05 A C
ATOM 571 CG LEU A 77 13, .050 8 .419 -2 .774 1, .00 11 .85 A C
ATOM 572 CD1 LEU A 77 12. .868 8, .361 -4, .281 1. .00 12 , .80 A C
ATOM 573 CD2 LEU A 77 12. .512 7, .140 -2, .114 1. .00 14, .07 A C
ATOM 574 C LEU A 77 15. .676 9, .847 -4, ,267 1. .00 13, .21 A C
ATOM 575 O LEU A 77 16, .181 8, .810 -4, .656 1. .00 13, .59 A O
ATOM 576 N GLY A 78 15, .586 10, .935 -5, .022 1. .00 13, .72 A N
ATOM 577 CA GLY A 78 16, .045 10, .945 -6, .415 1. .00 14, .19 A C
ATOM 578 C GLY A 78 17, .486 10 .505 -6, .506 1. .00 14 , .88 A C
ATOM 579 O GLY A 78 18. .322 10, .998 -5, .718 1. .00 14, .70 A O
ATOM 580 N ASN A 79 17, .800 9 .587 -7, .420 1. .00 15, .24 A N
ATOM 581 CA ASN A 79 19, ,172 9 .066 -7, .520 1. .00 16 .71 A C
ATOM 582 CB ASN A 79 19, .204 7 .542 -7, .263 1. .00 16, .08 A C
ATOM 583 CG ASN A 79 20. .615 7 .023 -6, .904 1. .00 16, .70 A C
ATOM 584 OD1 ASN A 79 21, .438 7 .754 -6, .372 1. .00 15, .21 A O
ATOM 585 ND2 ASN A 79 20, .881 5 .749 -7, .181 1. ,00 15, .92 A N
ATOM 586 C ASN A 79 19, .877 9 .353 -8, .852 1. .00 18, .12 A C
ATOM 587 O ASN A 79 20, .735 8 .576 -9, .267 1. .00 18, .96 A O
ATOM 588 N ALA A 80 19 .559 10 .458 -9. .513 1. ,00 18, .68 A N
ATOM 589 CA ALA A 80 20 .316 10 .838 -10, .723 1. .00 19, .08 A C
ATOM 590 CB ALA A 80 19 .381 11 .169 -11, .876 1. .00 19 .15 A C
ATOM 591 C ALA A 80 21 .261 11 .995 -10, .376 1. .00 18, .85 A C
ATOM 592 O ALA A 80 22 .245 11 .795 -9 .663 1. .00 18, .76 A O
ATOM 593 N THR A 81 20 .973 13 .194 -10, .841 1. .00 18, .39 A N
ATOM 594 CA THR A 81 21 .647 14 .370 -10, .305 1. ,00 18, .18 A C
ATOM 595 CB THR A 81 22, .229 15, .222 -11. .444 1. ,00 18. .72 A C
ATOM 596 OGl THR A 81 21. .202 15, .535 -12, .379 1. ,00 17. .48 A O
ATOM 597 CG2 THR A 81 23, ,229 14, .420 -12. .289 1. ,00 21. .52 A C
ATOM 598 C THR A 81 20. .650 15, .185 -9. .470 1. .00 17. .64 A C
ATOM 599 O THR A 81 19, .466 14 , .858 -9. .423 1. ,00 17. .77 A O
ATOM 600 N ASN A 82 21, .115 16, .238 -8. .803 1. ,00 16. .86 A N
ATOM 601 CA ASN A 82 20, .271 16 .947 -7. .842 1. ,00 16, .82 A C
ATOM 602 CB ASN A 82 19. .279 17 .840 -8, .574 1. .00 16. .90 A C
ATOM 603 CG ASN A 82 19, .962 18 .782 -9, .552 1. ,00 18. .05 A C
ATOM 604 OD1 ASN A 82 19, .861 18 .632 -10, .804 1. ,00 20, .14 A O
ATOM 605 ND2 ASN A 82 20 .650 19 .760 -9, .005 1. ,00 12 , .31 A N
ATOM 606 C ASN A 82 19, .541 15 .941 -6, .930 1. ,00 16, .16 A C
ATOM 607 O ASN A 82 18 .325 15 .985 -6, ,772 1. .00 16, .88 A O
ATOM 608 N LYS A 83 20 .310 15 .022 -6, .366 1. .00 15. .41 A N ATOM 609 CA LYS A 83 19,.767 13,.853 -5,.710 1,.00 15,.60 A C
ATOM 610 CB LYS A 83 20, .907 12 .919 -5, .287 1, .00 15 .95 A C
ATOM 611 CG LYS A 83 21, .665 12 .168 -6, ,415 1, .00 16 .19 A C
ATOM 612 CD LYS A 83 22, .815 11 .339 -5. .811 1, .00 19 .24 A C
ATOM 613 CE LYS A 83 23 .806 10 .791 -6, .833 1, .00 21 .12 A C
ATOM 614 NZ LYS A 83 23 .076 9 .941 -7, .791 1, .00 20 .88 A N
ATOM 615 C LYS A 83 18 .966 14 .243 -4, .453 1 .00 14 .74 A C
ATOM 616 O LYS A 83 19 .243 15 .248 -3, .801 1 .00 13 .71 A O
ATOM 617 N GLY A 84 18, .000 13, .402 -4. .117 1, .00 14 , .66 A N
ATOM 618 CA GLY A 84 17, .337 13, .439 -2, ,833 1, .00 14, .40 A C
ATOM 619 C GLY A 84 18, .240 13, .078 -1, .664 1, ,00 14 .38 A C
ATOM 620 O GLY A 84 19, .372 12, .683 -1, .853 1, .00 14 .68 A O
ATOM 621 N MET A 85 17, .734 13 .231 -0, ,439 1, .00 13 .41 A N
ATOM 622 CA MET A 85 18 .586 13 .079 0, ,753 1 .00 13 .39 A C
ATOM 623 CB MET A 85 17 .865 13 .660 1, .970 1 .00 13 .62 A C
ATOM 624 CG MET A 85 17 .446 15 .132 1, .799 1 .00 14 .10 A C
ATOM 625 SD MET A 85 18 .823 16 .235 1, .480 1 .00 15 .77 A S
ATOM 626 CE MET A 85 18 .801 16 .373 -0, .341 1 .00 16 .54 A C
ATOM 627 C MET A 85 18 .946 11 .600 1, .022 1 .00 13 .55 A C
ATOM 628 O MET A 85 19 .975 11 .302 1, .623 1 .00 13 .91 A O
ATOM 629 N ALA A 86 18, .078 10, .685 0, ,586 1, .00 13, .51 A N
ATOM 630 CA ALA A 86 18, .290 9 .250 0, .774 1, ,00 13, .69 A C
ATOM 631 CB ALA A 86 17 .223 8 .682 1, .717 1, ,00 13 .63 A C
ATOM 632 C ALA A 86 18, .200 8 .571 -0, ,589 1, .00 13 .59 A C
ATOM 633 O ALA A 86 17, .258 7 .821 -0, .868 1, .00 14 .50 A O
ATOM 634 N PRO A 87 19 .175 8 .818 -1, .445 1, .00 14 .33 A N
ATOM 635 CA PRO A 87 19 .068 8 .409 -2, .859 1, .00 14 .18 A C
ATOM 636 CB PRO A 87 20 .236 9 .152 -3. .515 1, .00 14 .93 A C
ATOM 637 CG PRO A 87 21 .263 9 .267 -2, .393 1, .00 14 .56 A C
ATOM 638 CD PRO A 87 20 .446 9 .524 -1, .148 1 .00 13 .97 A C
ATOM 639 C PRO A 87 19 .146 6 .901 -3. .123 1 .00 14 .68 A C
ATOM 640 O PRO A 87 18 .943 6 .474 -4 , .260 1 .00 15 .65 A O
ATOM 641 N GLN A 88 19. .424 6, .099 -2. ,109 1, .00 15, .11 A N
ATOM 642 CA GLN A 88 19, .436 4 , .639 -2. .266 1, ,00 16, .35 A C
ATOM 643 CB GLN A 88 20, .748 4 .050 -1. .733 1, .00 16, .73 A C
ATOM 644 CG GLN A 88 21, .900 4 .262 -2, .703 1, .00 19, .50 A C
ATOM 645 CD GLN A 88 23 .267 3 .916 -2, .161 1, .00 21, .39 A C
ATOM 646 OEl GLN A 88 23 .427 2 .933 -1, .439 1, .00 22 , .79 A O
ATOM 647 NE2 GLN A 88 24 .272 4 .709 -2, ,547 1, .00 22 .41 A N
ATOM 648 C GLN A 88 18 .228 3 .976 -1, .621 1, .00 16 .49 A C
ATOM 649 O GLN A 88 18 .080 2 .754 -1, .644 1, .00 16 .50 A O
ATOM 650 N ALA A 89 17 .347 4 .786 -1, .044 1, .00 16 .72 A N
ATOM 651 CA ALA A 89 16 .056 4 .279 -0, .599 1 .00 16 .78 A C
ATOM 652 CB ALA A 89 15, .380 5 .277 0. .375 1, ,00 17, .28 A C
ATOM 653 C ALA A 89 15, .139 3 .996 -1, .792 1, .00 16, .32 A C
ATOM 654 O ALA A 89 15, .212 4 .648 -2, .826 1, .00 16, .81 A O
ATOM 655 N ASN A 90 14, .248 3 .037 -1, .634 1, .00 15 .45 A N
ATOM 656 CA ASN A 90 13 .264 2 .756 -2, .658 1, .00 15 .40 A C
ATOM 657 CB ASN A 90 13 .036 1 .247 -2, .756 1, .00 16 .05 A C
ATOM 658 CG ASN A 90 14 .076 0 .549 -3, .658 1, .00 19 .92 A C
ATOM 659 OD1 ASN A 90 15 .039 1 .155 -4, ,106 1, .00 25 .00 A O
ATOM 660 ND2 ASN A 90 13 .892 -0 .736 -3, .873 1 .00 28 .28 A N
ATOM 661 C ASN A 90 11 .942 3 .486 -2, .367 1 .00 14 .01 A C
ATOM 662 O ASN A 90 11 .668 3 .834 -1, .234 1 .00 12 .81 A O
ATOM 663 N LEU A 91 11 .150 3 .705 -3, .410 1 .00 13 .02 A N
ATOM 664 CA LEU A 91 9, .964 4 .542 -3. .381 1, .00 13, .78 A C
ATOM 665 CB LEU A 91 10, .022 5 .524 -4 , .540 1, .00 13, .95 A C
ATOM 666 CG LEU A 91 8 .861 6 .472 -4, .765 1, .00 12 , .85 A C
ATOM 667 CD1 LEU A 91 8, .669 7 .375 -3, .571 1. .00 15 .80 A C
ATOM 668 CD2 LEU A 91 9 .077 7 .287 -6, .026 1. .00 13 .13 A C
ATOM 669 C LEU A 91 8 .661 3 .762 -3, .524 1, .00 14 .15 A C
ATOM 670 O LEU A 91 8 .503 2 .953 -4, .437 1, .00 15 .63 A O
ATOM 671 N VAL A 92 7 .716 4 .055 -2, .649 1, .00 13 .69 A N ATOM 672 CA VAL A 92 6.327 3.692 -2.872 1.00 14.00 A C
ATOM 673 CB VAL A 92 5 .737 3 .031 -1 .662 1 .00 13 .15 A C
ATOM 674 CGI VAL A 92 4 .197 3 .018 -1, .767 1 .00 15 .32 A C
ATOM 675 CG2 VAL A 92 6 .260 1 .621 -1 .546 1 .00 13 .02 A C
ATOM 676 C VAL A 92 5 .615 5 .001 -3 .175 1 .00 13 .45 A C
ATOM 677 O VAL A 92 5 .687 5 .942 -2 .376 1 .00 13 .53 A O
ATOM 678 N PHE A 93 4 .984 5 .107 -4 .346 1 .00 13 .19 A N
ATOM 679 CA PHE A 93 4 .293 6 .351 -4. .714 1 .00 13 .05 A C
ATOM 680 CB PHE A 93 4 .899 6 .964 -5 .985 1 .00 13 .57 A C
ATOM 681 CG PHE A 93 4 .484 8 .388 -6 .206 1 .00 13 .50 A C
ATOM 682 CD1 PHE A 93 5 .331 9 .439 -5 .861 1 .00 13 .00 A C
ATOM 683 CE1 PHE A 93 4 .941 10 .748 -6 .023 1 .00 12 .71 A C
ATOM 684 CZ PHE A 93 3 .680 11 .030 -6, .515 1 .00 11 .77 A C
ATOM 685 CE2 PHE A 93 2 .832 9 .998 -6 .881 1 .00 12 .22 A C
ATOM 686 CD2 PHE A 93 3 .226 8 .679 -6 .710 1 .00 12 .44 A C
ATOM 687 C PHE A 93 2 .793 6 .150 -4, .872 1 .00 13 .51 A C
ATOM 688 O PHE A 93 2 .350 5 .285 -5, .632 1 .00 13 .61 A O
ATOM 689 N GLN A 94 2 .021 6 .949 -4, .150 1 .00 13 .35 A N
ATOM 690 CA GLN A 94 0 .567 6 .903 -4, .197 1 .00 13 .55 A C
ATOM 691 CB GLN A 94 -0 .034 6 .775 -2, .786 1 .00 12 .95 A C
ATOM 692 CG GLN A 94 0 .383 5 .493 -2, .078 1 .00 13 .25 A C
ATOM 693 CD GLN A 94 0 .065 5 .494 -0, .589 1 .00 14 .07 A C
ATOM 694 OEl GLN A 94 0 .598 6 .311 0, .157 1 .00 16 .83 A O
ATOM 695 NE2 GLN A 94 -0 .813 4 .578 -0, .158 1 .00 13 .83 A N
ATOM 696 C GLN A 94 0 .118 8 .195 -4, .841 1 .00 13 .81 A C
ATOM 697 O GLN A 94 0 .197 9 .289 -4, .236 1 .00 12 .11 A O
ATOM 698 N SER A 95 -0 .266 8 .072 -6, .110 1 .00 14 .33 A N
ATOM 699 CA SER A 95 -0 .793 9 .190 -6, .893 1 .00 14 .08 A C
ATOM 700 CB SER A 95 -0 .743 8 .850 -8, .380 1 .00 13 .95 A C
ATOM 701 OG SER A 95 -1 .337 9 .864 -9, .152 1 .00 11 .92 A O
ATOM 702 C SER A 95 -2 .221 9 .519 -6, .494 1 .00 14 .82 A C
ATOM 703 O SER A 95 -3, .150 8 .743 -6, .780 1 .00 14 .73 A O
ATOM 704 N ILE A 96 -2, .404 10 .681 -5, .852 1 .00 15 .21 A N
ATOM 705 CA ILE A 96 -3, .699 11 .049 -5, .277 1 .00 15 .55 A C
ATOM 706 CB ILE A 96 -3, .560 11 .482 -3, .782 1 .00 15 .92 A C
ATOM 707 CGI ILE A 96 -2 .466 12 .548 -3, .597 1 .00 16 .08 A C
ATOM 708 CD1 ILE A 96 -2, .367 13 .122 -2, .196 1, .00 17 .02 A C
ATOM 709 CG2 ILE A 96 -3, .257 10 .273 -2, .915 1, .00 16 .70 A C
ATOM 710 C ILE A 96 -4, .398 12 .158 -6, .043 1, .00 15 .93 A C
ATOM 711 O ILE A 96 -5, .475 12 .590 -5. .660 1 .00 14 .84 A O
ATOM 712 N MET A 97 -3. .797 12 .640 -7, .119 1, .00 16 .32 A N
ATOM 713 CA MET A 97 -4, .440 13 .700 -7, ,889 1, .00 18 .17 A C
ATOM 714 CB MET A 97 -3, ,471 14 .346 -8. ,884 1, .00 18 .20 A C
ATOM 715 CG MET A 97 -4, ,107 15 .480 -9, .688 1, .00 21 .58 A C
ATOM 716 SD MET A 97 -2, .949 16 .297 -10, .814 1, .00 25 .41 A S
ATOM 717 CE MET A 97 -3, .900 16 .196 -12, .225 1, .00 31 .75 A C
ATOM 718 C MET A 97 -5, .647 13 .114 -8, ,641 1, .00 18 .32 A C
ATOM 719 O MET A 97 -5, .537 12 .054 -9. .249 1, .00 18 .26 A O
ATOM 720 N ASP A 98 -6, .780 13, .807 -8. .568 1, .00 19 .35 A N
ATOM 721 CA ASP A 98 -8. .020 13 .369 -9. .217 1, .00 20 .45 A C
ATOM 722 CB ASP A 98 -9, .268 13 .714 -8. .375 1, .00 20 .39 A C
ATOM 723 CG ASP A 98 -9, .367 15. .170 -8. .021 1, .00 21 .41 A C
ATOM 724 OD1 ASP A 98 -9, .234 16, .024 -8. .928 1, .00 22 .52 A O
ATOM 725 OD2 ASP A 98 -9. .599 15 .575 -6. .847 1. .00 20 .32 A O
ATOM 726 C ASP A 98 -8, ,093 14, .004 -10. ,592 1, .00 20 .49 A C
ATOM 727 O ASP A 98 -7, .168 14 .690 -10. .996 1, .00 19 .61 A O
ATOM 728 N SER A 99 -9, .170 13 .747 -11. .321 1, .00 21 .70 A N
ATOM 729 CA SER A 99 -9, .252 14, .202 -12. ,703 1. .00 23 .74 A C
ATOM 730 CB SER A 99 -10, ,202 13, .301 -13. .510 1, .00 24 .07 A C
ATOM 731 OG SER A 99 -11, .497 13 .436 -12. .986 1, .00 25 .19 A O
ATOM 732 C SER A 99 -9, .727 15 .660 -12. .749 1 .00 24 .80 A C
ATOM 733 O SER A 99 -9, .696 16 .286 -13, .812 1 .00 27 .46 A O
ATOM 734 N GLY A 100 -10, .152 16 .203 -11, .611 1, .00 24 .38 A N ATOM 735 CA GLY A 100 -10.425 17.621 -11.488 1.00 25.17 A C
ATOM 736 C GLY A 100 -9 .262 18 .470 -10 .968 1 .00 25 .36 A C
ATOM 737 O GLY A 100 -9 .475 19 .606 -10 .557 1 .00 26 .29 A O
ATOM 738 N GLY A 101 -8 .047 17 .933 -10 .964 1 .00 25 .13 A N
ATOM 739 CA GLY A 101 -6 .873 18 .715 -10 .573 1 .00 24 .90 A C
ATOM 740 C GLY A 101 -6 .541 18 .760 -9 .076 1 .00 24 .55 A C
ATOM 741 O GLY A 101 -5 .425 19 .133 -8 .713 1 .00 26 .15 A O
ATOM 742 N GLY A 102 -7 .490 18 .406 -8 .221 1 .00 22 .32 A N
ATOM 743 CA GLY A 102 -7 .258 18 .339 -6 .783 1 .00 21 .84 A C
ATOM 744 C GLY A 102 -6 .703 17 .008 -6 .267 1 .00 20 .73 A C
ATOM 745 O GLY A 102 -6 .172 16 .204 -7 .021 1 .00 19 .11 A O
ATOM 746 N LEU A 103 -6 .814 16 .794 -4 .959 1 .00 19 .97 A N
ATOM 747 CA LEU A 103 -6 .225 15 .634 -4 .294 1 .00 19 .19 A C
ATOM 748 CB LEU A 103 -5 .346 16 .094 -3 .131 1 .00 18 .87 A C
ATOM 749 CG LEU A 103 -4 .169 16 .986 -3 .552 1 .00 18 .31 A C
ATOM 750 CD1 LEU A 103 -3 .298 17 .397 -2 .354 1 .00 17 .54 A C
ATOM 751 CD2 LEU A 103 -3 .341 16 .297 -4 .607 1 .00 19 .64 A C
ATOM 752 C LEU A 103 -7 .307 14 .676 -3 .809 1 .00 19 .31 A C
ATOM 753 O LEU A 103 -7 .179 14 .018 -2 .750 1 .00 18 .44 A O
ATOM 754 N GLY A 104 -8, .371 14 .586 -4 .604 1 .00 18 .93 A N
ATOM 755 CA GLY A 104 -9, .537 13 .780 -4 .260 1 .00 18 .78 A C
ATOM 756 C GLY A 104 -9, .259 12 .298 -4. .234 1, .00 18 .26 A C
ATOM 757 O GLY A 104 -10, .078 11 .506 -3 .780 1, .00 19 .17 A O
ATOM 758 N GLY A 105 -8, .094 11 .886 -4. .703 1, .00 17 .54 A N
ATOM 759 CA GLY A 105 -7, .698 10 .500 -4. .520 1, .00 17 .23 A C
ATOM 760 C GLY A 105 -7, .395 10 .091 -3, , 075 1, .00 16 .85 A C
ATOM 761 O GLY A 105 -7, .319 8, .895 -2, .731 1, .00 15 .88 A O
ATOM 762 N LEU A 106 -7, .263 11, .067 -2, .194 1, .00 16 .62 A N
ATOM 763 CA LEU A 106 -7, .137 10, .729 -0, ,777 1, .00 16 .79 A C
ATOM 764 CB LEU A 106 -6, .892 11, .975 0. .048 1. .00 16 .05 A C
ATOM 765 CG LEU A 106 -5, ,519 12, .560 -0. .204 1. ,00 14 .68 A C
ATOM 766 CD1 LEU A 106 -5. .479 13 , .986 0. .274 1. .00 16 .20 A C
ATOM 767 CD2 LEU A 106 -4. .425 11, .707 0. .507 1. .00 13 .15 A C
ATOM 768 C LEU A 106 -8. .423 10, .056 -0. .304 1. ,00 17 .90 A C
ATOM 769 O LEU A 106 -9, ,513 10 , ,553 -0. .587 1. .00 18 .63 A O
ATOM 770 N PRO A 107 -8. .318 8, ,932 0, ,387 1. .00 18 .42 A N
ATOM 771 CA PRO A 107 -9. .506 8, .280 0. ,977 1. .00 19 .35 A C
ATOM 772 CB PRO A 107 -8. ,963 6, .932 1. .430 1. .00 19, .15 A C
ATOM 773 CG PRO A 107 -7. .537 7 , ,286 1. ,774 1. .00 19, .19 A C
ATOM 774 CD PRO A 107 -7. .089 8. .162 0. .640 1. ,00 18, .63 A C
ATOM 775 C PRO A 107 -10, ,070 9, .036 2. , 178 1. .00 18. .88 A C
ATOM 776 O PRO A 107 -9. ,340 9, .724 2. ,886 1. .00 19. .33 A O
ATOM 777 N ALA A 108 -11, .367 8, .910 2. ,408 1, ,00 19, ,46 A N
ATOM 778 CA ALA A 108 -12, .022 9, .562 3. .530 1. .00 19, .50 A C
ATOM 779 CB ALA A 108 -13, .514 9, .168 3. .585 1. .00 20, .76 A C
ATOM 780 C ALA A 108 -11. .359 9, .229 4. ,875 1. ,00 18, .72 A C
ATOM 781 O ALA A 108 -11. .229 10, .093 5. .727 1. ,00 19, .17 A O
ATOM 782 N ASN A 109 -11. ,007 7 , .964 5. ,069 1. ,00 18. .94 A N
ATOM 783 CA ASN A 109 -10. .193 7 , .535 6. ,209 1. 00 19. .10 A C
ATOM 784 CB ASN A 109 -10. .691 6, .206 6. ,773 1. ,00 19. .41 A C
ATOM 785 CG ASN A 109 -9. ,990 5, .834 8. ,073 1. 00 22. .66 A C
ATOM 786 OD1 ASN A 109 -8. .872 6, .295 8. ,349 1. 00 19. .31 A O
ATOM 787 ND2 ASN A 109 -10. ,665 5. .018 8. ,908 1. 00 25. .73 A N
ATOM 788 C ASN A 109 -8. .731 7. .392 5. ,804 1. 00 17. .97 A C
ATOM 789 O ASN A 109 -8. .353 6. .446 5. ,088 1. 00 17. ,31 A O
ATOM 790 N LEU A 110 -7. .895 8. .325 6. ,245 1. 00 16. ,66 A N
ATOM 791 CA LEU A 110 -6. .489 8. .277 5. ,862 1. 00 15. ,50 A C
ATOM 792 CB LEU A 110 -5. .738 9. .502 6. ,406 1. 00 15. ,65 A C
ATOM 793 CG LEU A 110 -6. .096 10. .831 5. ,749 1. 00 13. .74 A C
ATOM 794 CD1 LEU A 110 -5. .294 11. .932 6. ,373 1. 00 16. ,06 A C
ATOM 795 CD2 LEU A 110 -5. .873 10. .768 4. ,256 1. 00 13. ,53 A C
ATOM 796 C LEU A 110 -5. .784 7. .006 6. ,285 1. 00 15. ,96 A C
ATOM 797 O LEU A 110 -4 , .750 6. .660 5. ,719 1. 00 16. ,04 A O ATOM 798 N GLN A 111 -6.297 6.283 7.276 1.00 16.18 A N
ATOM 799 CA GLN A 111 -5, .635 5 .034 7 .655 1 .00 16 .61 A C
ATOM 800 CB GLN A 111 -6 .317 4 .377 8 .871 1 .00 17 .82 A C
ATOM 801 CG GLN A 111 -6 .337 5 .320 10, .077 1 .00 17 .25 A C
ATOM 802 CD GLN A 111 -6, .584 4 .625 11, .399 1 .00 20 .40 A C
ATOM 803 OEl GLN A 111 -5 .934 3 .635 11, .699 1 .00 21 .99 A O
ATOM 804 NE2 GLN A 111 -7 .513 5 .163 12, .202 1 .00 19 .23 A N
ATOM 805 C GLN A 111 -5, .560 4 .086 6, .461 1 .00 17 .45 A C
ATOM 806 O GLN A 111 -4 .601 3 .323 6, .312 1 .00 17 .74 A O
ATOM 807 N THR A 112 -6, .522 4 .195 5, .548 1 .00 16 .47 A N
ATOM 808 CA THR A 112 -6 .483 3 .418 4, .309 1 .00 16 .12 A C
ATOM 809 CB THR A 112 -7 .756 3 .733 3, .510 1 .00 16 .21 A C
ATOM 810 OGl THR A 112 -8 .900 3 .480 4, .333 1 .00 17 .07 A O
ATOM 811 CG2 THR A 112 -7 .909 2 .838 2, .305 1 .00 16 .70 A C
ATOM 812 C THR A 112 -5 .252 3 .711 3 .442 1 .00 15 .86 A C
ATOM 813 O THR A 112 -4 .623 2 .789 2 .869 1 .00 15 .50 A O
ATOM 814 N LEU A 113 -4 .933 4 .995 3 .303 1 .00 14 .75 A N
ATOM 815 CA LEU A 113 -3 .742 5 .413 2 .558 1 .00 14 .05 A C
ATOM 816 CB LEU A 113 -3 .677 6 .941 2 .557 1 .00 14 .27 A C
ATOM 817 CG LEU A 113 -2 .549 7 .597 1 .807 1 .00 14 .53 A C
ATOM 818 CD1 LEU A 113 -2 .840 7 .473 0 .297 1 .00 16 .65 A C
ATOM 819 CD2 LEU A 113 -2 .412 9 .039 2 .212 1 .00 13 .95 A C
ATOM 820 C LEU A 113 -2 .478 4 .836 3 .212 1 .00 13 .77 A C
ATOM 821 O LEU A 113 -1 .625 4 .238 2 .550 1 .00 13 .55 A O
ATOM 822 N PHE A 114 -2 .361 5 .016 4 .523 1 .00 12 .95 A N
ATOM 823 CA PHE A 114 -1 .182 4 .528 5, .223 1 .00 13 .04 A C
ATOM 824 CB PHE A 114 -1 .154 5 .049 6, .645 1 .00 12 .56 A C
ATOM 825 CG PHE A 114 -1 .331 6 .551 6, .743 1 .00 11 .79 A C
ATOM 826 CD1 PHE A 114 -0 .639 7 .402 5, .902 1 .00 12 .07 A C
ATOM 827 CEl PHE A 114 -0 .785 8 .781 5, .986 1 .00 12 .47 A C
ATOM 828 CZ PHE A 114 -1 .662 9 .323 6, .921 1 .00 13 .57 A C
ATOM 829 CE2 PHE A 114 -2 .365 8 .470 7, .754 1 .00 11 .94 A C
ATOM 830 CD2 PHE A 114 -2 .186 7 .100 7, ,663 1 .00 9 .85 A C
ATOM 831 C PHE A 114 -1 .060 3 .003 5, .171 1 .00 13 .86 A C
ATOM 832 O PHE A 114 0 .063 2 .461 5, .004 1 .00 12 .73 A O
ATOM 833 N SER A 115 -2 .196 2 .306 5, .277 1 .00 14 .04 A N
ATOM 834 CA SER A 115 -2 .148 0 .848 5, .292 1. .00 14. .17 A C
ATOM 835 CB : BSER A 115 -3 .527 0 .252 5, ,640 0, .50 13. .81 A C
ATOM 836 CB ; /.SER A 115 -3 .457 0 .215 5, .769 0, .50 14. .55 A C
ATOM 837 OG : BSER A 115 -3 .970 0 .566 6, .958 0, .50 10, .51 A O
ATOM 838 OG . J.SER A 115 -4 .544 0 .608 4, .978 0, .50 18, .03 A O
ATOM 839 c SER A 115 -1 .677 0 .296 3, ,943 1, .00 14 , .11 A C
ATOM 840 0 SER A 115 -0 .932 -0 .663 3, .909 1, .00 13, .43 A O
ATOM 841 N GLN A 116 -2 .108 0 .890 2, .832 1, .00 14, .73 A N
ATOM 842 CA GLN A 116 -1. .656 0 .442 1, ,513 1, ,00 14, .53 A C
ATOM 843 CB GLN A 116 -2 .394 1 .234 0. .417 1, .00 15, .81 A C
ATOM 844 CG GLN A 116 -1 .947 0 .951 -1, .038 1, .00 15, .88 A C
ATOM 845 CD GLN A 116 -2 .601 1 .886 -2, .007 1, ,00 16. .75 A C
ATOM 846 OEl GLN A 116 -2 .629 3, .086 -1. .747 1, .00 14. ,56 A O
ATOM 847 NE2 GLN A 116 -3 .200 1 .346 -3. .106 1, .00 14. .07 A N
ATOM 848 C GLN A 116 -0 .131 0 .571 1. .375 1, .00 14. .16 A C
ATOM 849 O GLN A 116 0 .554 -0 .336 0. .861 1, .00 14. .37 A O
ATOM 850 N ALA A 117 0 .407 1 .679 1. .862 1. .00 13. .34 A N
ATOM 851 CA ALA A 117 1 .838 1 .930 1, .795 1. .00 13. .79 A C
ATOM 852 CB ALA A 117 2 .152 3, .408 2. ,151 1. .00 13. .52 A C
ATOM 853 C ALA A 117 2 .608 0, .972 2. .714 1. .00 13. .17 A C
ATOM 854 O ALA A 117 3 .666 0, .472 2. ,344 1. .00 13. .07 A O
ATOM 855 N TYR A 118 2 .071 0, .740 3. .908 1. ,00 13. .32 A N
ATOM 856 CA TYR A 118 2 .679 -0, .161 4. .877 1. .00 13. ,82 A C
ATOM 857 CB TYR A 118 1 .878 -0, .177 6. .190 1. ,00 14. ,02 A C
ATOM 858 CG TYR A 118 2 .636 -0, .861 7. .324 1. .00 17. ,04 A C
ATOM 859 CD1 TYR A 118 2 .472 -2 .216 7, ,589 1, ,00 20. .14 A C
ATOM 860 CEl TYR A 118 3 .186 -2 .839 8, .640 1, ,00 24. .14 A C ATOM 861 CZ TYR A 118 4,.041 -2.071 9,.409 1,.00 23.61 A C
ATOM 862 OH TYR A 118 4, .762 -2 .631 10, .442 1, .00 28 .49 A O
ATOM 863 CE2 TYR A 118 4, .194 -0 .725 9, .155 1, .00 20 .69 A C
ATOM 864 CD2 TYR A 118 3 .501 -0 .135 8 .136 1 .00 18 .61 A C
ATOM 865 C TYR A 118 2 .782 -1 .576 4 .294 1 .00 14 .18 A C
ATOM 866 O TYR A 118 3, .838 -2 .228 4 .363 1 .00 14 .05 A O
ATOM 867 N SER A 119 1 .705 -2 .024 3 .669 1 .00 14 .24 A N
ATOM 868 CA SER A 119 1. .684 -3 .358 3 .064 1 .00 15 .13 A C
ATOM 869 CB SER A 119 0 .288 -3 .660 2 .544 1 .00 14 .77 A C
ATOM 870 OG SER A 119 -0 .609 -3 .744 3 .638 1 .00 13 .56 A O
ATOM 871 C SER A 119 2 .752 -3 .531 1 .977 1 .00 15 .67 A C
ATOM 872 O SER A 119 3 .313 -4 .602 1 .818 1 .00 15 .80 A O
ATOM 873 N ALA A 120 3 .052 -2 .461 1 .254 1 .00 16 .24 A N
ATOM 874 CA ALA A 120 4 .085 -2 .488 0 .204 1 .00 15 .93 A C
ATOM 875 CB ALA A 120 3 .847 -1 .352 -0 .759 1 .00 16 .16 A C
ATOM 876 C ALA A 120 5 .504 -2 .405 0 .767 1 .00 16 .13 A c
ATOM 877 O ALA A 120 6 .474 -2 .473 0 .030 1 .00 16 .93 A 0
ATOM 878 N GLY A 121 5 .626 -2 .249 2 .083 1 .00 16 .23 A N
ATOM 879 CA GLY A 121 6, .917 -2 .249 2, .747 1, .00 15 .35 A c
ATOM 880 C GLY A 121 7, .400 -0 .883 3, .247 1, .00 15 .15 A c
ATOM 881 O GLY A 121 8 .466 -0 .811 3, .893 1, .00 15 .57 A O
ATOM 882 N ALA A 122 6, .665 0 .195 2, .977 1, .00 14 .40 A N
ATOM 883 CA ALA A 122 7. .110 1 .522 3, .443 1, .00 14 .85 A C
ATOM 884 CB ALA A 122 6 .273 2 .632 2, .831 1, ,00 14 .84 A C
ATOM 885 C ALA A 122 7 .057 1 .635 4, .964 1, .00 14 .35 A C
ATOM 886 O ALA A 122 6, .078 1 .230 5, .574 1, .00 15 .21 A O
ATOM 887 N ARG A 123 8 .077 2 .223 5 .570 1 .00 13 .62 A N
ATOM 888 CA ARG A 123 8 .013 2 .499 7 .008 1 .00 12 .85 A C
ATOM 889 CB ARG A 123 9 .065 1 .689 7 .761 1 .00 12 .85 A C
ATOM 890 CG ARG A 123 8 .870 0 .162 7 .597 1, .00 13 .74 A C
ATOM 891 CD ARG A 123 7 .584 -0 .334 8 .290 1, .00 14 .35 A C
ATOM 892 NE ARG A 123 7 .396 -1 .786 8 .187 1, .00 14 .77 A N
ATOM 893 CZ ARG A 123 6 .676 -2 .389 7 .253 1, .00 16 .63 A C
ATOM 894 NHl ARG A 123 6 .039 -1 .678 6 .337 1, .00 15 .68 A N
ATOM 895 NH2 ARG A 123 6 .579 -3 .719 7 .240 1, .00 17 .36 A N
ATOM 896 C ARG A 123 8 .132 3 .987 7 .298 1, .00 12 .72 A C
ATOM 897 O ARG A 123 8 .116 4 .418 8, .448 1, .00 12 .15 A O
ATOM 898 N ILE A 124 8 .225 4 .773 6, .234 1, .00 12 .67 A N
ATOM 899 CA ILE A 124 8 .177 6 .218 6, .346 1, .00 12 .97 A C
ATOM 900 CB ILE A 124 9 .554 6 .814 6, .025 1, ,00 12 .64 A C
ATOM 901 CGI ILE A 124 10 .619 6 .262 6, .985 1, .00 13, .71 A C
ATOM 902 CD1 ILE A 124 12 .068 6 .395 6, ,480 1. .00 14, .82 A C
ATOM 903 CG2 ILE A 124 9 .478 8 .348 6, .061 1. ,00 13, .97 A C
ATOM 904 C ILE A 124 7 .160 6 .695 5, .324 1. .00 12 , .91 A C
ATOM 905 O ILE A 124 7 .132 6 .195 4, .210 1. .00 13. .07 A O
ATOM 906 N HIS A 125 6 .365 7 .696 5, .671 1. ,00 12. .94 A N
ATOM 907 CA HIS A 125 5 .252 8 .100 4, .823 1. .00 12, ,77 A C
ATOM 908 CB HIS A 125 3 .894 7 .549 5. .353 1. .00 13, .00 A C
ATOM 909 CG HIS A 125 2, .806 7 .650 4. ,334 1. .00 15, .91 A C
ATOM 910 NDl HIS A 125 2 .428 8 .850 3, .783 1, .00 13, .47 A N
ATOM 911 CEl HIS A 125 1 .547 8 .632 2, .821 1, .00 16, .40 A C
ATOM 912 NE2 HIS A 125 1 .312 7 .333 2, .756 1, .00 16, .49 A N
ATOM 913 CD2 HIS A 125 2 .072 6 .699 3, .705 1. ,00 18, .35 A C
ATOM 914 C HIS A 125 5 .223 9 .620 4, .828 1. .00 12, .62 A C
ATOM 915 O HIS A 125 5 .053 10 .202 5, ,893 1. .00 12, .04 A O
ATOM 916 N THR A 126 5 .401 10 .268 3. .674 1. .00 12, .71 A N
ATOM 917 CA THR A 126 5 .527 11 .738 3. .641 1. .00 12, .63 A C
ATOM 918 CB THR A 126 6 .984 12 .142 3, .302 1. .00 12 , .63 A C
ATOM 919 OGl THR A 126 7 .121 13 .560 3, .334 1. ,00 12. .18 A O
ATOM 920 CG2 THR A 126 7 .395 11 .747 1, .864 1. ,00 12. .36 A C
ATOM 921 C THR A 126 4 .498 12 .426 2 , .735 1. ,00 12. .60 A C
ATOM 922 O THR A 126 4 .166 11 .931 1. .652 1. ,00 12. .62 A O
ATOM 923 N ASN A 127 4 .010 13 .572 3, .200 1. .00 12. .52 A N ATOM 924 CA ASN A 127 2.778 14.189 .696 1.00 12.84 A C
ATOM 925 CB ASN A 127 1 .599 13 .811 3 .605 1 .00 13 .04 A C
ATOM 926 CG ASN A 127 1 .433 12 .325 3, .720 1 .00 13 .25 A C
ATOM 927 OD1 ASN A 127 1 .916 11 .686 4 .690 1 .00 13 .15 A O
ATOM 928 ND2 ASN A 127 0 .814 11 .740 .712 1 .00 9 .82 A N
ATOM 929 C ASN A 127 2 .894 15 .706 2 .637 1 .00 12 .70 A C
ATOM 930 O ASN A 127 2 .798 16 .390 3 .661 1 .00 13 .27 A O
ATOM 931 N SER A 128 3 .103 16 .211 1 .435 1 .00 12 .76 A N
ATOM 932 CA SER A 128 3 .277 17 .640 1 .162 1 .00 13 .02 A C
ATOM 933 CB SER A 128 4 .308 17 .831 0. ,043 1 .00 12 .57 A C
ATOM 934 OG SER A 128 5 .608 17 .510 0 .485 1 .00 12 .52 A O
ATOM 935 C SER A 128 1 .927 18 .238 0 .748 1 .00 13 .42 A C
ATOM 936 O SER A 128 1 .763 18 .767 -0 .372 1 .00 13 .82 A O
ATOM 937 N TRP A 129 0 .968 18 .129 1, .663 1 .00 13 .86 A N
ATOM 938 CA TRP A 129 -0 .392 18 .616 1 .465 1 .00 13 .67 A C
ATOM 939 CB TRP A 129 -1 .215 17 .648 0, .602 1 .00 13 .88 A C
ATOM 940 CG TRP A 129 -1 .130 16 .180 0 .964 1 .00 13 .08 A C
ATOM 941 CD1 TRP A 129 -0 .305 15 .232 0, .391 1 .00 14 .80 A C
ATOM 942 NE1 TRP A 129 -0 .531 13 .997 0 .956 1 .00 12 .40 A N
ATOM 943 CE2 TRP A 129 -1 .518 14 .122 1, .900 1 .00 13 .40 A C
ATOM 944 CD2 TRP A 129 -1 .924 15 .480 1, .921 1 .00 12 .64 A C
ATOM 945 CE3 TRP A 129 -2 .948 15 .857 2, .806 1 .00 14 .15 A C
ATOM 946 CZ3 TRP A 129 -3 .504 14 .910 3, .614 1, .00 13 .25 A C
ATOM 947 CH2 TRP A 129 -3 .082 13 .566 3, ,559 1 .00 13 .78 A C
ATOM 948 CZ2 TRP A 129 -2 .101 13 .158 2, .711 1, .00 12 .56 A C
ATOM 949 C TRP A 129 -1. .089 18 .859 2. .782 1, .00 14 .44 A C
ATOM 950 O TRP A 129 -0 .612 18 .460 3, .876 1, .00 14 .19 A O
ATOM 951 N GLY A 130 -2 .224 19 .538 2, .694 1 .00 14 .75 A N
ATOM 952 CA GLY A 130 -3 .004 19 .834 3, .866 1, .00 15 .45 A C
ATOM 953 C GLY A 130 -4. .173 20 .744 3. .563 1, ,00 16 .66 A C
ATOM 954 O GLY A 130 -4 .203 21 .394 2, ,518 1, ,00 16 .04 A O
ATOM 955 N ALA A 131 -5 .139 20 .754 4, .478 1 .00 16 .87 A N
ATOM 956 CA ALA A 131 -6. .222 21, .733 4. .484 1. .00 18, .19 A C
ATOM 957 CB ALA A 131 -7, .515 21 .097 4. ,983 1, ,00 17 .33 A C
ATOM 958 C ALA A 131 -5 .843 22 .852 5. .423 1, .00 19 .54 A C
ATOM 959 O ALA A 131 -5. .562 22, .590 6. ,592 1, ,00 20 .18 A O
ATOM 960 N PRO A 132 -5. .869 24 .090 4. .942 1, .00 20, .97 A N
ATOM 961 CA PRO A 132 -5. .513 25 .253 5. .763 1, ,00 21 .46 A C
ATOM 962 CB PRO A 132 -5 .260 26 .346 4. .724 1, .00 21 .95 A C
ATOM 963 CG PRO A 132 -6. .060 25, .967 3. .546 1. .00 22 .18 A C
ATOM 964 CD PRO A 132 -6, .220 24 .462 3. .564 1. .00 21 .43 A C
ATOM 965 C PRO A 132 -6 .595 25 .676 6, .753 1, .00 22 .74 A C
ATOM 966 O PRO A 132 -7, ,272 26 .703 6. .555 1, .00 24 .13 A O
ATOM 967 N VAL A 133 -6, .708 24, .912 7, .833 1. .00 22, .71 A N
ATOM 968 CA VAL A 133 -7, .723 25 .086 8. .850 1, .00 23 .39 A C
ATOM 969 CB VAL A 133 -8, .349 23, .712 9. .223 1. .00 23, .48 A C
ATOM 970 CGI VAL A 133 -9, ,115 23 .133 8. .045 1, ,00 25 .68 A C
ATOM 971 CG2 VAL A 133 -7. .269 22, .750 9. .687 1. .00 24, .53 A C
ATOM 972 C VAL A 133 -7. .223 25 .742 10. .150 1, .00 23 .23 A C
ATOM 973 O VAL A 133 -7. ,855 25, .599 11. ,185 1. ,00 22, .57 A O
ATOM 974 N ASN A 134 -6. .094 26 .437 10. .098 1, .00 23 .10 A N
ATOM 975 CA ASN A 134 -5. .660 27, .279 11. ,201 1. .00 23, .36 A C
ATOM 976 CB ASN A 134 -6. ,583 28, .512 11. ,310 1. ,00 24, ,40 A C
ATOM 977 CG ASN A 134 -6. .491 29, .413 10. ,082 1. .00 26, .68 A C
ATOM 978 OD1 ASN A 134 -7. ,489 30. .000 9. .650 1, .00 34 .40 A O
ATOM 979 ND2 ASN A 134 -5. ,315 29. .478 9. 482 1. ,00 28. .87 A N
ATOM 980 C ASN A 134 -5. ,588 26. .561 12. ,535 1, ,00 22, .43 A C
ATOM 981 O ASN A 134 -6. .210 26 .971 13. .510 1. .00 21. .30 A O
ATOM 982 N GLY A 135 -4. .844 25, .458 12. ,574 1. ,00 21. .06 A N
ATOM 983 CA GLY A 135 -4. ,548 24, .840 13. ,846 1. ,00 20, ,36 A C
ATOM 984 C GLY A 135 -5, .541 23 .818 14. ,308 1. .00 19, .66 A C
ATOM 985 O GLY A 135 -5, .320 23 .200 15. .327 1, .00 18 .95 A O
ATOM 986 N ALA A 136 -6. ,613 23. .595 13. 557 1. ,00 19. .08 A N ATOM 987 CA ALA A 136 -7.609 22.643 14,.006 1.00 19.00 A C
ATOM 988 CB ALA A 136 -8 .925 22 .778 13 .199 1 .00 19 .34 A C
ATOM 989 C ALA A 136 -7 .098 21 .206 13 .893 1 .00 19 .31 A C
ATOM 990 O ALA A 136 -6 .354 20 .851 12 .952 1 .00 18 .44 A O
ATOM 991 N TYR A 137 -7 .568 20 .407 14 .841 1 .00 18 .56 A N
ATOM 992 CA TYR A 137 -7 .341 18 .979 14 .907 1 .00 18 .99 A C
ATOM 993 CB TYR A 137 -7 .112 18 .588 16 .367 1 .00 18 .67 A C
ATOM 994 CG TYR A 137 -6 .637 17 .175 16 .588 1 .00 19 .68 A C
ATOM 995 CDl TYR A 137 -7 .537 16 .173 16 .885 1 .00 19 .55 A c
ATOM 996 CEl TYR A 137 -7 .112 14 .855 17 .099 1 .00 21 .07 A c
ATOM 997 CZ TYR A 137 -5 .765 14 .548 17 .045 1 .00 20 .92 A c
ATOM 998 OH TYR A 137 -5 .371 13 .250 17 .265 1 .00 20 .20 A 0
ATOM 999 CE2 TYR A 137 -4 .837 15 .538 16 .754 1 .00 20 .35 A c
ATOM 1000 CD2 TYR A 137 -5 .278 16 .848 16, .522 1 .00 19 .75 A c
ATOM 1001 C TYR A 137 -8 .600 18 .314 14, .337 1 .00 18 .91 A c
ATOM 1002 O TYR A 137 -9 .648 18 .229 14, .994 1 .00 18 .41 A 0
ATOM 1003 N THR A 138 -8 .481 17 .872 13, .091 1 .00 18 .62 A N
ATOM 1004 CA THR A 138 -9 .608 17 .401 12, .329 1 .00 17 .86 A c
ATOM 1005 CB THR A 138 -9, .480 17 .836 10, .897 1 .00 18 .02 A C
ATOM 1006 OGl THR A 138 -8, .271 17 .308 10, ,321 1 .00 16 .63 A 0
ATOM 1007 CG2 THR A 138 -9, .308 19 .330 10, .788 1. .00 17 .63 A c
ATOM 1008 C THR A 138 -9, .593 15 .888 12. .407 1 .00 18, .43 A c
ATOM 1009 O THR A 138 -8, .662 15 .296 12. .954 1 .00 17 .82 A 0
ATOM 1010 N THR A 139 10, .624 15 .278 11. .843 1 .00 18, .06 A N
ATOM 1011 CA THR A 139 10, .713 13 .843 11. .705 1 .00 17, .69 A c
ATOM 1012 CB THR A 139 12. .020 13 .472 10. .947 1, .00 18, .57 A C
ATOM 1013 OGl THR A 139 13 .162 13 .907 11, .705 1 .00 20 .05 A O
ATOM 1014 CG2 THR A 139 12 .173 11 .933 10, .828 1 .00 19 .16 A C
ATOM 1015 C THR A 139 -9. .496 13 .285 10, .989 1. .00 16 .78 A c
ATOM 1016 O THR A 139 -9, .037 12 .183 11, .307 1 .00 17 .05 A 0
ATOM 1017 N ASP A 140 -8, .976 14 .002 10, .002 1, .00 15, .81 A N
ATOM 1018 CA ASP A 140 -7, .758 13 .544 9, .351 1 .00 15, .65 A C
ATOM 1019 CB ASP A 140 -7, ,391 14 .429 8. .177 1 .00 15, .45 A C
ATOM 1020 CG ASP A 140 -8, .279 14 .209 6. .984 1 .00 16, .65 A C
ATOM 1021 ODl ASP A 140 -8, .495 15 .189 6. ,263 1 .00 18, .76 A O
ATOM 1022 OD2 ASP A 140 -8, .781 13, .102 6. .702 1, .00 16, .74 A O
ATOM 1023 C ASP A 140 -6. .567 13, .504 10. .352 1, .00 15, .55 A c
ATOM 1024 O ASP A 140 -5. .823 12, .532 10. ,395 1, .00 15, ,72 A 0
ATOM 1025 N SER A 141 -6. ,395 14 , .555 11. ,133 1, .00 15, .34 A N
ATOM 1026 CA SER A 141 -5. ,375 14 , ,548 12. ,187 1, .00 15 , ,53 A C
ATOM 1027 CB SER A 141 -5. .428 15, ,823 13. ,006 1, .00 14, ,57 A C
ATOM 1028 OG SER A 141 -5. .275 16, .936 12. ,173 1, .00 16, .14 A O
ATOM 1029 C SER A 141 -5. ,514 13 , .375 13. ,157 1, .00 15 , .46 A C
ATOM 1030 0 SER A 141 -4. .511 12 , .754 13. ,558 1, .00 15, .37 A O
ATOM 1031 N ARG A 142 -6. .754 13 , .100 13. ,546 1, .00 15, .27 A N
ATOM 1032 CA ARG A 142 -7. ,053 11, .998 14. ,462 1 , ,00 15. .97 A C
ATOM 1033 CB ARG A 142 -8. .539 12 , .004 14. 843 1 , ,00 16. .78 A C
ATOM 1034 CG ARG A 142 -8. .882 11. ,091 16. 022 1. .00 18. .90 A C
ATOM 1035 CD ARG A 142 10. ,365 11. .103 16. 436 1. .00 22. .40 A C
ATOM 1036 NE ARG A 142 10. .533 10, ,384 17. ,704 1, .00 25, .70 A N
ATOM 1037 CZ ARG A 142 10. .549 9, .057 17. ,839 1, .00 29, ,38 A C
ATOM 1038 NHl ARG A 142 10. .423 8, .249 16. ,786 1, .00 30, .26 A N
ATOM 1039 NH2 ARG A 142 10. .685 8, .524 19. ,048 1, .00 30, .56 A N
ATOM 1040 C ARG A 142 -6. .703 10, .643 13. ,860 1, .00 15, ,43 A C
ATOM 1041 O ARG A 142 -6. ,107 9. .778 14. ,534 1, ,00 14. .36 A O
ATOM 1042 N ASN A 143 -7. ,068 10, .437 12. ,593 1, .00 14 , .96 A N
ATOM 1043 CA ASN A 143 -6. .699 9, .187 11. ,926 1, .00 14. .70 A C
ATOM 1044 CB ASN A 143 -7. .451 9, .062 10. ,593 1 , .00 15. .54 A C
ATOM 1045 CG ASN A 143 -8. ,952 8. .709 10. ,803 1. .00 16. .85 A C
ATOM 1046 ODl ASN A 143 -9. ,842 9. .204 10. ,096 1. ,00 20. .66 A O
ATOM 1047 ND2 ASN A 143 -9. ,206 7. .828 11. ,746 1. .00 15. .73 A N
ATOM 1048 C ASN A 143 -5. ,183 8. ,986 11. ,754 1, .00 15. .26 A C
ATOM 1049 O ASN A 143 -4. ,691 7. .854 11. ,879 1 , .00 15. ,15 A 0 ATOM 1050 N VAL A 144 -4.438 10.060 11,.450 1..00 14,.90 A N
ATOM 1051 CA VAL A 144 -2 .976 9, .987 11, .467 1, .00 13, .93 A C
ATOM 1052 CB VAL A 144 -2 .319 11 .347 11, .177 1, ,00 14 , .08 A C
ATOM 1053 CGI VAL A 144 -0 .803 11 .272 11. .422 1, .00 12, .32 A C
ATOM 1054 CG2 VAL A 144 -2 .625 11 .818 9. .748 1, ,00 13 .08 A C
ATOM 1055 C VAL A 144 -2 .478 9 .507 12. .843 1, .00 14 .23 A C
ATOM 1056 O VAL A 144 -1 .608 8 .653 12. .938 1, .00 13 .96 A O
ATOM 1057 N ASP A 145 -3 .021 10 .077 13. .916 1, .00 14 .48 A N
ATOM 1058 CA ASP A 145 -2, .548 9. .745 15, .256 1. .00 14 , .49 A C
ATOM 1059 CB ASP A 145 -3 .123 10, .711 16, .249 1. ,00 14 , ,81 A C
ATOM 1060 CG ASP A 145 -2 .406 12 , .033 16, .218 1. .00 15, .70 A C
ATOM 1061 ODl ASP A 145 -1 .332 12, .107 15, .545 1, .00 14 , .69 A O
ATOM 1062 OD2 ASP A 145 -2 .845 13, .048 16, ,803 1, .00 14, .46 A O
ATOM 1063 C ASP A 145 -2 .849 8, .331 15, .654 1, .00 15, .08 A C
ATOM 1064 O ASP A 145 -1 .999 7 .622 16, .183 1. .00 14, .89 A O
ATOM 1065 N ASP A 146 -4 .065 7 .906 15, .361 1, .00 15, .66 A N
ATOM 1066 CA ASP A 146 -4 .470 6 .545 15, .608 1, .00 15, .98 A C
ATOM 1067 CB ASP A 146 -5 .931 6 .400 15. .184 1, .00 16, .37 A C
ATOM 1068 CG ASP A 146 -6 .565 5 .107 15. .705 1, .00 17, .81 A C
ATOM 1069 ODl ASP A 146 -6 .337 4 .735 16, .879 1, .00 18 .17 A O
ATOM 1070 OD2 ASP A 146 -7, .277 4 , ,401 14. .981 1. ,00 21, .66 A O
ATOM 1071 C ASP A 146 -3, .562 5, .571 14. .849 1. .00 16, .07 A C
ATOM 1072 O ASP A 146 -3, .047 4 , .607 15, .408 1. ,00 16, .60 A O
ATOM 1073 N TYR A 147 -3, .324 5, .842 13. .576 1. .00 15, .73 A N
ATOM 1074 CA TYR A 147 -2 .463 4 , .988 12, .772 1. .00 15 , ,49 A C
ATOM 1075 CB TYR A 147 -2 .387 5. .486 11, ,314 1. .00 15. .22 A C
ATOM 1076 CG TYR A 147 -1 .759 4 .421 10, .459 1, .00 16. .31 A C
ATOM 1077 CDl TYR A 147 -0 .400 4 .394 10. ,249 1, .00 17, .35 A C
ATOM 1078 CEl TYR A 147 0 .180 3 .380 9. ,506 1, .00 17, .49 A C
ATOM 1079 CZ TYR A 147 -0 .599 2 .364 9, , 004 1, .00 16, .46 A C
ATOM 1080 OH TYR A 147 -0 .022 1 .354 8, .281 1, .00 20, .80 A O
ATOM 1081 CE2 TYR A 147 -1 .944 2 .346 9, .227 1, .00 16, .19 A C
ATOM 1082 CD2 TYR A 147 -2, ,523 3, .364 9. ,947 1. .00 16. .98 A C
ATOM 1083 C TYR A 147 -1, .025 4 , .833 13. .309 1. ,00 15. .68 A C
ATOM 1084 O TYR A 147 -0, .491 3, .719 13, .385 1. .00 14, .79 A O
ATOM 1085 N VAL A 148 -0 .399 5, .953 13, ,652 1. .00 16, .33 A N
ATOM 1086 CA VAL A 148 0 .975 5, .950 14, .144 1. ,00 16, .18 A C
ATOM 1087 CB VAL A 148 1 .534 7 .390 14, .262 1. .00 16, .37 A C
ATOM 1088 CGI VAL A 148 2 .953 7 .397 14, ,909 1, .00 17, .53 A C
ATOM 1089 CG2 VAL A 148 1 .600 8 .044 12, .899 1, .00 16, .39 A C
ATOM 1090 C VAL A 148 1 .063 5 .206 15, .488 1, .00 16 .59 A C
ATOM 1091 O VAL A 148 2 .022 4 .481 15, .765 1, .00 16 .63 A O
ATOM 1092 N ARG A 149 0 .061 5 .356 16 .331 1, .00 16 .70 A N
ATOM 1093 CA ARG A 149 0, .109 4 .628 17, .589 1. .00 18, .28 A C
ATOM 1094 CB ARG A 149 -0 .920 5 .133 18, .600 1. .00 18, .33 A C
ATOM 1095 CG ARG A 149 -0 .585 4 .657 20, .002 1, ,00 19, .51 A C
ATOM 1096 CD ARG A 149 -1 .566 5 .035 21, .071 1, .00 20 .84 A C
ATOM 1097 NE ARG A 149 -0 .987 4 .731 22, .383 1, .00 22 .92 A N
ATOM 1098 CZ ARG A 149 -1 .661 4 .491 23, .504 1, .00 24 .06 A C
ATOM 1099 NHl ARG A 149 -2 .985 4 .521 23, .538 1. .00 25 .69 A N
ATOM 1100 NH2 ARG A 149 -0 .987 4 .221 24, .616 1, .00 23 .61 A N
ATOM 1101 C ARG A 149 -0 .035 3 .126 17 .382 1, .00 18 .63 A C
ATOM 1102 O ARG A 149 0 .517 2 .346 18 .156 1, .00 18 .97 A O
ATOM 1103 N LYS A 150 -0 .739 2 .720 16 .326 1 .00 18 .98 A N
ATOM 1104 CA LYS A 150 -0 .991 1 .294 16 .087 1 .00 19 .24 A C
ATOM 1105 CB LYS A 150 -2 .373 1 .092 15, .438 1, ,00 19 .89 A C
ATOM 1106 CG LYS A 150 -3 .576 1 .358 16, .389 1, ,00 21 .34 A C
ATOM 1107 CD LYS A 150 -4 .902 0 .972 15, .736 1, .00 24 .20 A C
ATOM 1108 CE LYS A 150 -6 .136 1 .437 16, .531 1, .00 27 .20 A C
ATOM 1109 NZ LYS A 150 -7 .373 1 .614 15 .668 1, .00 30 .36 A N
ATOM 1110 C L"ΫS A 150 0 .123 0 .622 15 .250 1 .00 18 .99 A C
ATOM 1111 O LYS A 150 0 .296 -0 .577 15 .305 1 .00 17 .16 A O
ATOM 1112 N ASN A 151 0 .916 1 .407 14 .526 1 .00 19 .09 A N ATOM 1113 CA ASN A 151 1,.834 0.850 13,.538 1.00 19.75 A C
ATOM 1114 CB ASN A 151 1, ,225 0, .950 12, .130 1 .00 20 .10 A C
ATOM 1115 CG ASN A 151 -0, .141 0, .299 12, .025 1 .00 19 .86 A C
ATOM 1116 ODl ASN A 151 -0, .239 -0, .905 11. .855 1 .00 19 .33 A O
ATOM 1117 ND2 ASN A 151 -1, ,198 1 .090 12. .167 1 .00 19 .31 A N
ATOM 1118 C ASN A 151 3. .150 1 .599 13, .557 1 .00 20 .21 A C
ATOM 1119 O ASN A 151 3, .193 2, .807 13, .793 1 .00 22 .14 A O
ATOM 1120 N ASP A 152 4, .239 0, .911 13. .299 1 .00 20 .04 A N
ATOM 1121 CA ASP A 152 5, .508 1 .595 13. .319 1 .00 20 .51 A C
ATOM 1122 CB ] BASP A 152 6. .571 0 .640 13, .830 0 .35 20 .16 A C
ATOM 1123 CB i Z-ASP A 152 6. .645 0 .666 13, .762 0 .65 21 .56 A C
ATOM 1124 CG ! BASP A 152 6, .199 0 .067 15. .205 0 .35 19 .09 A C
ATOM 1125 CG ; A&SP A 152 7, .225 -0 .117 12. .631 0 .65 23 .76 A C
ATOM 1126 OD1BASP A 152 5. .318 0 .654 15. ,901 0 .35 15 .06 A O
ATOM 1127 OD1AASP A 152 6, .404 -0 .719 11, .924 0 .65 27 .77 A O
ATOM 1128 OD2BASP A 152 6, .703 -0 .977 15, .653 0 .35 16 .81 A O
ATOM 1129 OD2AASP A 152 8, .471 -0 .170 12, .353 0, .65 27 .40 A O
ATOM 1130 C ASP A 152 5, .822 2 .270 11, .959 1, .00 19 .32 A C
ATOM 1131 O ASP A 152 6, .748 1, .916 11, .253 1, .00 20 .60 A O
ATOM 1132 N MET A 153 4, .988 3, .250 11, .628 1, .00 16 .58 A N
ATOM 1133 CA MET A 153 5. .154 4 .050 10, ,437 1, .00 16 .03 A C
ATOM 1134 CB MET A 153 3, .876 4 , .007 9, ,619 1, .00 16 .23 A C
ATOM 1135 CG MET A 153 3, .885 4 .921 8. .432 1, .00 18 .33 A C
ATOM 1136 SD MET A 153 4 , .694 4 .182 7, .030 1, .00 21 .72 A S
ATOM 1137 CE MET A 153 3. .290 3 .549 6, ,297 1, .00 21 .74 A C
ATOM 1138 C MET A 153 5, .443 5, .482 10, .871 1, .00 14 .80 A C
ATOM 1139 O MET A 153 4, .684 6, .058 11, ,646 1, .00 13 .50 A O
ATOM 1140 N THR A 154 6, .525 6, .059 10, .368 1, .00 13 .34 A N
ATOM 1141 CA THR A 154 6, ,813 7, .482 10. .638 1, .00 13 .08 A C
ATOM 1142 CB THR A 154 8. .324 7, .699 10. ,652 1, ,00 13 .02 A C
ATOM 1143 OGl THR A 154 8, .886 6, .949 11. .724 1, .00 11 .51 A O
ATOM 1144 CG2 THR A 154 8, ,693 9, .145 10. .963 1. .00 14 .36 A C
ATOM 1145 C THR A 154 6. .153 8, .310 9, ,573 1, .00 12 .50 A C
ATOM 1146 O THR A 154 6, .396 8, .108 8. .371 1, .00 12 .64 A O
ATOM 1147 N ILE A 155 5, .290 9 .227 9, .987 1, .00 12 .34 A N
ATOM 1148 CA ILE A 155 4, .492 10 .002 9. .037 1, .00 13 .00 A C
ATOM 1149 CB ILE A 155 2, .983 9 .799 9, ,346 1, .00 13 .05 A C
ATOM 1150 CGI ILE A 155 2, .637 8 .307 9. .279 1, .00 13 .73 A C
ATOM 1151 CDl ILE A 155 1, .121 8 .017 9. .274 1. .00 12 .93 A C
ATOM 1152 CG2 ILE A 155 2, .121 10 .578 8, ,371 1, .00 13 .58 A C
ATOM 1153 C ILE A 155 4 .861 11 .480 9. .137 1, .00 13 .01 A C
ATOM 1154 O ILE A 155 4 .894 12 .038 10, .233 1, .00 12 .79 A 0
ATOM 1155 N LEU A 156 5 .125 12 .117 8, .001 1, .00 12 .16 A N
ATOM 1156 CA LEU A 156 5 .509 13 .528 7, .982 1, .00 12 .88 A C
ATOM 1157 CB LEU A 156 6 .903 13 .692 7, .354 1, .00 12 .18 A C
ATOM 1158 CG LEU A 156 8 .089 12 .960 8, .007 1, .00 13 .25 A C
ATOM 1159 CDl LEU A 156 8 .365 11 .607 7, .326 1 .00 11 .24 A C
ATOM 1160 CD2 LEU A 156 9 .339 13 .796 7, .910 1, .00 13 .01 A C
ATOM 1161 C LEU A 156 4 .485 14 .328 7, .192 1 .00 13 .49 A C
ATOM 1162 O LEU A 156 3 .982 13 .850 6, .160 1, .00 14 .76 A O
ATOM 1163 N PHE A 157 4 .197 15 .540 7, .659 1 .00 12 .79 A N
ATOM 1164 CA PHE A 157 3 .282 16 .451 7, .003 1, .00 13 .07 A C
ATOM 1165 CB PHE A 157 1 .938 16 .564 7, .772 1, .00 13 .62 A C
ATOM 1166 CG PHE A 157 0 .957 15 .504 7, .401 1, .00 13 .90 A C
ATOM 1167 CDl PHE A 157 0 .191 15 .636 6, .272 1, .00 11 .22 A C
ATOM 1168 CEl PHE A 157 -0 .678 14 .632 5, .896 1, .00 14 .22 A C
ATOM 1169 CZ PHE A 157 -0 .743 13 .441 6, .630 1 .00 14 .61 A C
ATOM 1170 CE2 PHE A 157 0 .013 13 .296 7, .743 1, .00 15 .64 A C
ATOM 1171 CD2 PHE A 157 0 .891 14 .312 8, .122 1, .00 14 .56 A C
ATOM 1172 C PHE A 157 3 .899 17 .852 6, .928 1, .00 13 .18 A C
ATOM 1173 O PHE A 157 4 .527 18 .318 7 .867 1 .00 12 .04 A 0
ATOM 1174 N ALA A 158 3 .700 18 .500 5 .793 1 .00 13 .46 A N
ATOM 1175 CA ALA A 158 3 .958 19 .921 5 .623 1 .00 13 .31 A C ATOM 1176 CB ALA A 158 3,.509 20.334 4,.235 1..00 14.02 A C
ATOM 1177 C ALA A 158 3, .181 20 .703 6, .672 1, .00 13 .75 A C
ATOM 1178 O ALA A 158 2 , ,031 20 .380 6, ,965 1, .00 13 .75 A O
ATOM 1179 N ALA A 159 3, .787 21 .752 7, .215 1, .00 13 .45 A N
ATOM 1180 CA ALA A 159 3, .122 22 .582 8. .210 1, .00 13 .81 A C
ATOM 1181 CB ALA A 159 4 .151 23 .495 8, .944 1, .00 13 .64 A C
ATOM 1182 C ALA A 159 2 .043 23 .473 7, .628 1, .00 14 .14 A C
ATOM 1183 O ALA A 159 1 .175 23 .924 8, .364 1, ,00 14 .00 A O
ATOM 1184 N GLY A 160 2 .131 23 .753 6, .330 1, .00 15 .19 A N
ATOM 1185 CA GLY A 160 1 .230 24 .680 5, .652 1, .00 15 .34 A C
ATOM 1186 C GLY A 160 1 .957 25 .941 5, .236 1, .00 14 .93 A C
ATOM 1187 O GLY A 160 3 .041 26 .238 5, .736 1, .00 14 .62 A O
ATOM 1188 N ASN A 161 1 .371 26 .686 4, .307 1, .00 15 .04 A N
ATOM 1189 CA ASN A 161 1 .983 27 .902 3, .789 1, .00 15 .84 A C
ATOM 1190 CB ASN A 161 2, .072 27 .872 2, .261 1. .00 15 .91 A C
ATOM 1191 CG ASN A 161 3, .048 26, .851 1, .712 1. .00 17 .40 A C
ATOM 1192 ODl ASN A 161 3 , ,001 26, .550 0, ,490 1. .00 21 .70 A O
ATOM 1193 ND2 ASN A 161 3, .888 26 .267 2, .569 1, .00 11 .15 A N
ATOM 1194 C ASN A 161 1, .131 29 .114 4 , .123 1, .00 16 .84 A C
ATOM 1195 O ASN A 161 0, .956 29 .965 3, .286 1, .00 17 .07 A O
ATOM 1196 N GLU A 162 0, .575 29 .179 5, .324 1, .00 18 .36 A N
ATOM 1197 CA GLU A 162 -0, .392 30 .213 5, .668 1, ,00 18 .85 A C
ATOM 1198 CB GLU A 162 -1 .672 29 .537 6, .211 1, .00 19 .64 A C
ATOM 1199 CG GLU A 162 -2 .431 28 .723 5, .150 1, .00 22 .12 A C
ATOM 1200 CD GLU A 162 -1 .756 27 .381 4 , .788 1, ,00 26 .12 A C
ATOM 1201 OEl GLU A 162 -1 .585 26 .545 5, .702 1, .00 28 .48 A O
ATOM 1202 OE2 GLU A 162 -1, .405 27 .149 3, .590 1, .00 26 .75 A O
ATOM 1203 C GLU A 162 0 .147 31 .262 6, .657 1, .00 19 .39 A C
ATOM 1204 O GLU A 162 -0 .633 32 .031 7, .225 1, .00 18 .80 A O
ATOM 1205 N GLY A 163 1 .472 31 .338 6, .820 1, .00 19 .39 A N
ATOM 1206 CA GLY A 163 2 , ,082 32, .322 7. ,705 1. .00 20, .03 A C
ATOM 1207 C GLY A 163 2, .224 33, .699 7, .048 1. ,00 21, .41 A C
ATOM 1208 O GLY A 163 1, .822 33 .866 5, ,877 1. .00 20, .72 A O
ATOM 1209 N PRO A 164 2, .835 34 .671 7, .737 1, .00 21 .93 A N
ATOM 1210 CA PRO A 164 3, .496 34 .491 9. .053 1. .00 22 .22 A C
ATOM 1211 CB PRO A 164 4, ,575 35 .577 9, ,050 1, .00 22 .99 A C
ATOM 1212 CG PRO A 164 3, .945 36 .720 8. .171 1, ,00 23 .13 A C
ATOM 1213 CD PRO A 164 2, .976 36 .047 7, .209 1, .00 22 .03 A C
ATOM 1214 C PRO A 164 2, .681 34 .621 10, .329 1, .00 22 .18 A C
ATOM 1215 O PRO A 164 3, .289 34 .603 11, .414 1, .00 21 .36 A O
ATOM 1216 N GLY A 165 1. .363 34 .702 10, .239 1, .00 21 .66 A N
ATOM 1217 CA GLY A 165 0 .537 34 .844 11, .414 1, .00 21 .84 A C
ATOM 1218 C GLY A 165 0 .522 33 .581 12, .243 1, .00 22 .27 A C
ATOM 1219 O GLY A 165 0 .680 32 .440 11. .713 1, ,00 22 .20 A O
ATOM 1220 N SER A 166 0 .305 33 .762 13. .543 1, .00 22 .06 A N
ATOM 1221 CA SER A 166 0, ,290 32, .645 14, .470 1. .00 21, .96 A C
ATOM 1222 CB SER A 166 0. .344 33, .167 15. .917 1. ,00 23 ,25 A C
ATOM 1223 OG SER A 166 -0, .948 33 .579 16. .367 1. .00 25, .13 A O
ATOM 1224 C SER A 166 -0, .954 31 .807 14. .241 1. .00 21 .45 A C
ATOM 1225 O SER A 166 -1. .949 32 .311 13, .716 1. .00 21 .15 A O
ATOM 1226 N GLY A 167 -0, .879 30 .515 14, .574 1, .00 20 .07 A N
ATOM 1227 CA GLY A 167 -2, .032 29 .639 14, ,548 1, .00 19 .63 A C
ATOM 1228 C GLY A 167 -2, .478 29 .248 13, .140 1, .00 19 .18 A C
ATOM 1229 O GLY A 167 -3, .652 29 .051 12, .911 1, .00 18 .57 A O
ATOM 1230 N THR A 168 -1, .541 29 .140 12, .200 1, .00 18 .31 A N
ATOM 1231 CA THR A 168 -1 .893 28 .857 10, .810 1, .00 17 .13 A C
ATOM 1232 CB THR A 168 -1 .295 29 .958 9, .908 1, .00 17 .32 A C
ATOM 1233 OGl THR A 168 0 .077 30 .172 10. .261 1, ,00 14 .44 A O
ATOM 1234 CG2 THR A 168 -1 .988 31 .299 10. .156 1, .00 17 .60 A C
ATOM 1235 C THR A 168 -1 .496 27 .465 10 .306 1. .00 16 .75 A C
ATOM 1236 O THR A 168 -1 .462 27 .213 9 .091 1 .00 16 .26 A O
ATOM 1237 N ILE A 169 -1, .265 26 .540 11, .234 1, .00 15 .95 A N
ATOM 1238 CA ILE A 169 -0, .863 25 .191 10, .871 1, .00 15 .48 A C ATOM 1239 CB ILE A 169 0.454 24.378 12.127 1.00 15.09 A C
ATOM 1240 CGI ILE A 169 0 .626 25 .109 12 .942 1 .00 14 .88 A C
ATOM 1241 CDl ILE A 169 2 .021 25 .201 12 .267 1 .00 16 .38 A C
ATOM 1242 CG2 ILE A 169 0 .021 22 .988 11 .720 1. .00 13 .45 A C
ATOM 1243 C ILE A 169 2 .004 24, .477 10 .137 1 .00 15 .22 A C
ATOM 1244 O ILE A 169 3, .146 24 .470 10, .590 1 .00 14 .50 A O
ATOM 1245 N SER A 170 1, .681 23 .857 9, .018 1 .00 14 .94 A N
ATOM 1246 CA SER A 170 2, .665 23, .060 8, .310 1 .00 15 .41 A C
ATOM 1247 CB SER A 170 2, .299 23 .004 6, .821 1 .00 16 .02 A C
ATOM 1248 OG SER A 170 1, .040 22, .404 6, .585 1 .00 16 .03 A O
ATOM 1249 C SER A 170 2, .855 21, .660 8, .904 1 .00 15 .05 A C
ATOM 1250 O SER A 170 1, .986 21 .137 9, .616 1 .00 13 .80 A O
ATOM 1251 N ALA A 171 3, .992 21, .036 8, .582 1 .00 14 .65 A N
ATOM 1252 CA ALA A 171 4, .244 19, .651 8, .933 1 .00 14 .75 A C
ATOM 1253 CB ALA A 171 5 .700 19 .507 9 .443 1 .00 15 .62 A C
ATOM 1254 C ALA A 171 4 .043 18 .750 7 .740 1 .00 15 .02 A C
ATOM 1255 O ALA A 171 4 .475 19 .096 6 .652 1 .00 14 .12 A O
ATOM 1256 N PRO A 172 3 .482 17 .548 7 .899 1 .00 15 .48 A N
ATOM 1257 CA PRO A 172 3 .078 16 .913 9 .167 1 .00 15 .17 A C
ATOM 1258 CB PRO A 172 3 .080 15 .411 8 .796 1 .00 15 .90 A C
ATOM 1259 CG PRO A 172 3 .707 15 .336 7 .456 1 .00 15 .73 A C
ATOM 1260 CD PRO A 172 3 .401 16 .614 6 .768 1 .00 15 .43 A C
ATOM 1261 C PRO A 172 1 .724 17 .260 9 .788 1 .00 14 .85 A C
ATOM 1262 O PRO A 172 1 .284 16 .499 10 .651 1 .00 16 .20 A O
ATOM 1263 N GLY A 173 1 .086 18 .352 9 .382 1 .00 14 .01 A N
ATOM 1264 CA GLY A 173 0 .078 18 .895 10 .064 1 .00 13 .48 A C
ATOM 1265 C GLY A 173 0 .158 19 .147 11 .553 1 .00 13 .91 A C
ATOM 1266 O GLY A 173 0 .809 19 .174 12 .339 1 .00 14 .08 A O
ATOM 1267 N THR A 174 1 .419 19 .291 11 .956 1 .00 13 .50 A N
ATOM 1268 CA THR A 174 1 .758 19 .475 13, .375 1 .00 13 .19 A C
ATOM 1269 CB THR A 174 3 .137 20 .125 13, .530 1 .00 13 .66 A C
ATOM 1270 OGl THR A 174 4 .104 19 .394 12 .743 1 .00 13 .16 A O
ATOM 1271 CG2 THR A 174 3 .114 21 .510 12 .957 1 .00 14 .22 A C
ATOM 1272 C THR A 174 1 .774 18 .188 14, .172 1 .00 12 .85 A C
ATOM 1273 O THR A 174 1 .909 18 .227 15 ,390 1 .00 12 .08 A O
ATOM 1274 N ALA A 175 1 .696 17 .040 13, .505 1 .00 12 .94 A N
ATOM 1275 CA ALA A 175 1 .614 15 .772 14, .213 1 .00 12 .90 A C
ATOM 1276 CB ALA A 175 1 .422 14 .641 13, .211 1 .00 13 .54 A C
ATOM 1277 C ALA A 175 0 .484 15 .740 15, .264 1, .00 12 .44 A C
ATOM 1278 O ALA A 175 0 .601 16 .233 15, .043 1 .00 13 .06 A O
ATOM 1279 N LYS A 176 0 .739 15 .131 16, .398 1, .00 13 .08 A N
ATOM 1280 CA LYS A 176 0 .269 15 .057 17, .466 1, .00 13 .00 A C
ATOM 1281 CB LYS A 176 0 .383 14 .511 18. .719 1, .00 12 .78 A C
ATOM 1282 CG LYS A 176 1 .406 15 .392 19, .366 1, .00 13 .87 A C
ATOM 1283 CD LYS A 176 2 .044 14 .693 20, .553 1, .00 15 .77 A C
ATOM 1284 CE LYS A 176 3, .179 13 .722 20, .173 1, .00 16 .63 A C
ATOM 1285 NZ LYS A 176 3, .738 13 .048 21, .388 1, .00 16 .58 A N
ATOM 1286 C LYS A 176 1, .433 14 .107 17, ,115 1. .00 13, .10 A C
ATOM 1287 O LYS A 176 2, .559 14 .289 17, .538 1, ,00 12 , .98 A O
ATOM 1288 N ASN A 177 1, .119 13 .047 16, .390 1, .00 12 , .81 A N
ATOM 1289 CA ASN A 177 2, .047 11, .933 16. .187 1. .00 12, .78 A C
ATOM 1290 CB ASN A 177 1, ,278 10, .628 16. .301 1, .00 12 , .48 A C
ATOM 1291 CG ASN A 177 0, .733 10, .382 17. .718 1. .00 12 , .12 A C
ATOM 1292 ODl ASN A 177 1, .135 11, .043 18. ,682 1. ,00 12 , .31 A O
ATOM 1293 ND2 ASN A 177 0, ,179 9, .416 17. .844 1. ,00 10, .81 A N
ATOM 1294 C ASN A 177 2, .822 11, .966 14. .876 1. ,00 12. .68 A C
ATOM 1295 O ASN A 177 3, .692 11, .097 14. .621 1. ,00 13. .33 A O
ATOM 1296 N ALA A 178 2, .483 12, .933 14. ,029 1. ,00 12. .80 A N
ATOM 1297 CA ALA A 178 3, .234 13, .208 12. ,801 1. ,00 12. .70 A C
ATOM 1298 CB ALA A 178 2, ,382 13, .938 11. ,817 1. ,00 12. .83 A C
ATOM 1299 C ALA A 178 4 , .439 14 , .052 13. ,141 1. ,00 12. .18 A C
ATOM 1300 O ALA A 178 4 .471 14 .685 14. .188 1. .00 12 , .29 A O
ATOM 1301 N ILE A 179 5 .458 13 .985 12. .293 1. .00 11, .71 A N ATOM 1302 CA ILE A 179 6,.531 14.966 12.283 1.00 11.72 A C
ATOM 1303 CB ILE A 179 7, .838 14 .364 11 .812 1 .00 11 .54 A C
ATOM 1304 CGI ILE A 179 8, .251 13 .222 12 .712 1 .00 13 .45 A C
ATOM 1305 CDl ILE A 179 9, .472 12 .467 12, .196 1 .00 14 .82 A C
ATOM 1306 CG2 ILE A 179 8, .927 15 .437 11 .783 1 .00 12 .00 A C
ATOM 1307 C ILE A 179 6, .085 16 .076 11 .317 1 .00 11 .98 A C
ATOM 1308 O ILE A 179 5. .943 15 .852 10 ,109 1 .00 11 .31 A O
ATOM 1309 N THR A 180 5, .813 17 .248 11 .871 1 .00 11 .27 A N
ATOM 1310 CA THR A 180 5, .357 18 .383 11 .074 1 .00 11 .76 A C
ATOM 1311 CB THR A 180 4, .260 19 .120 11 .818 1 .00 11 .79 A C
ATOM 1312 OGl THR A 180 3, .166 18 .214 12 .084 1 .00 12 .06 A O
ATOM 1313 CG2 THR A 180 3 .603 20 .224 10 .929 1 .00 12 .72 A C
ATOM 1314 C THR A 180 6 .530 19 .306 10 .690 1 .00 11 .82 A C
ATOM 1315 O THR A 180 7 .286 19 .762 11 .533 1 .00 11 .02 A O
ATOM 1316 N VAL A 181 6, .662 19 .590 9, .401 1, .00 11 .42 A N
ATOM 1317 CA VAL A 181 7 , .830 20 .305 8, .899 1, .00 11 .43 A C
ATOM 1318 CB VAL A 181 8, .492 19 .464 7, .814 1, .00 11 .32 A C
ATOM 1319 CGI VAL A 181 9, .744 20, .118 7, .309 1, .00 12, .42 A C
ATOM 1320 CG2 VAL A 181 8, .757 18 .055 8, .351 1, .00 12, .33 A C
ATOM 1321 C VAL A 181 7, .511 21 .680 8, ,302 1, .00 11 .62 A C
ATOM 1322 O VAL A 181 6, .667 21 .800 7, ,399 1, .00 12, .16 A O
ATOM 1323 N GLY A 182 8, .187 22 .704 8, .812 1, .00 11 .59 A N
ATOM 1324 CA GLY A 182 8, .095 24 .042 8, ,273 1, .00 12 .80 A C
ATOM 1325 C GLY A 182 9, .296 24 .352 7, .391 1, .00 13 .72 A C
ATOM 1326 O GLY A 182 10, .243 23 .574 7, .344 1, .00 14 .13 A O
ATOM 1327 N ALA A 183 9, .264 25 .492 6, .700 1, .00 13 .43 A N
ATOM 1328 CA ALA A 183 10, .312 25 .837 5, .776 1, .00 14 .22 A C
ATOM 1329 CB ALA A 183 9, .709 26 .166 4, .401 1, .00 14 .53 A C
ATOM 1330 C ALA A 183 11, .205 27 .001 6, ,238 1, .00 14 .32 A C
ATOM 1331 O ALA A 183 10, .717 28 .110 6, .498 1, .00 14 .13 A O
ATOM 1332 N THR A 184 12, .512 26 .737 6, .293 1, .00 14 .17 A N
ATOM 1333 CA THR A 184 13, .513 27 .799 6, ,294 1, .00 14 .33 A C
ATOM 1334 CB THR A 184 14, .743 27 .451 7, ,159 1, .00 14 .19 A C
ATOM 1335 OGl THR A 184 15, .180 26 .103 6, .925 1, .00 13 .83 A O
ATOM 1336 CG2 THR A 184 14, .383 27 .474 8, .636 1, .00 13 .70 A C
ATOM 1337 C THR A 184 13, .905 28 .018 4, .841 1, .00 15 .47 A C
ATOM 1338 O THR A 184 13. .380 27 .354 3. .934 1, .00 15 .87 A O
ATOM 1339 N GLU A 185 14. .861 28 .919 4, .618 1 .00 15 .13 A N
ATOM 1340 CA GLU A 185 15, .328 29 .246 3. ,290 1, .00 14 .03 A C
ATOM 1341 CB GLU A 185 15, .696 30 .766 3, .230 1 .00 13 .82 A C
ATOM 1342 CG GLU A 185 14, .492 31 .673 3, .495 1 .00 15 .09 A C
ATOM 1343 CD GLU A 185 14, .785 33 .172 3, .329 1 .00 14 .09 A C
ATOM 1344 OEl GLU A 185 15, .911 33 .541 2, .985 1 .00 15 .60 A O
ATOM 1345 OE2 GLU A 185 13, .871 33 .984 3, .528 1 .00 14 .21 A O
ATOM 1346 C GLU A 185 16, .511 28 .376 2, .863 1 .00 13 .70 A C
ATOM 1347 O GLU A 185 17, .387 28 .011 3, ,675 1 .00 14 .17 A O
ATOM 1348 N ASN A 186 16. .521 28 .008 1, .587 1 .00 12 .64 A N
ATOM 1349 CA ASN A 186 17. .707 27 .452 0, ,959 1, .00 13 .44 A C
ATOM 1350 CB ASN A 186 17, .345 26 .758 -0, .353 1 .00 13 .84 A C
ATOM 1351 CG ASN A 186 18, .293 25 .630 -0, .717 1 .00 14 .82 A C
ATOM 1352 ODl ASN A 186 19, .084 25 .169 0, .099 1 .00 14 .62 A O
ATOM 1353 ND2 ASN A 186 18, .189 25 .156 -1, .970 1 .00 15 .51 A N
ATOM 1354 C ASN A 186 18, .652 28 .603 0, .681 1 .00 13 .83 A C
ATOM 1355 O ASN A 186 18, .244 29 .769 0, ,711 1 .00 14 .50 A O
ATOM 1356 N LEU A 187 19, .920 28 .298 0, .470 1, .00 14 .83 A N
ATOM 1357 CA LEU A 187 20, .892 29 .352 0, .213 1, .00 14 .62 A C
ATOM 1358 CB LEU A 187 22, .144 29 .144 1, .018 1 .00 15 .48 A C
ATOM 1359 CG LEU A 187 23, .144 30 .319 0, .975 1, .00 17 .25 A C
ATOM 1360 CDl LEU A 187 22, .504 31 .587 1, .469 1 .00 18 .56 A C
ATOM 1361 CD2 LEU A 187 24, .394 29 .973 1. .816 1 .00 20 .46 A C
ATOM 1362 C LEU A 187 21 .205 29 .360 -1, .279 1 .00 14 .80 A C
ATOM 1363 O LEU A 187 22, .106 28, .692 -1. ,734 1, .00 14, .07 A O
ATOM 1364 N ARG A 188 20, .398 30, .083 -2. ,023 1, .00 15, .63 A N ATOM 1365 CA ARG A 188 20.631 30.308 3.454 1.00 17.55 A C
ATOM 1366 CB ARG A 188 19 .658 29 .484 4 .273 1 .00 17 .02 A C
ATOM 1367 CG ARG A 188 19 .842 27 .989 4 .168 1 .00 17 .82 A C
ATOM 1368 CD ARG A 188 19 .063 27 .213 5 .267 1. .00 19 .96 A C
ATOM 1369 NE ARG A 188 19 .315 25 .782 5 .224 1 .00 18 .26 A N
ATOM 1370 CZ ARG A 188 20 .339 25 .172 5 .814 1 .00 19 .52 A C
ATOM 1371 NHl ARG A 188 21 .235 25 .846 6 .530 1 .00 17 .91 A N
ATOM 1372 NH2 ARG A 188 20 .475 23 .867 5 .693 1 .00 19 .41 A N
ATOM 1373 C ARG A 188 20 .387 31 .804 3 .694 1. .00 17 .89 A C
ATOM 1374 O ARG A 188 19 .379 32 .189 4 .251 1 .00 18 .33 A O
ATOM 1375 N PRO A 189 21 .273 32 .646 3 .181 1. .00 19 .58 A N
ATOM 1376 CA PRO A 189 20 .990 34 .082 3 .061 1 .00 20 .68 A C
ATOM 1377 CB PRO A 189 22 .179 34 .613 2 .239 1 .00 21 .07 A C
ATOM 1378 CG PRO A 189 23 .271 33 .608 2 .417 1. .00 21 .15 A C
ATOM 1379 CD PRO A 189 22 .599 32 .288 2 .657 1. .00 20 .12 A C
ATOM 1380 C PRO A 189 20 .833 34 .863 4 .373 1 .00 21 .39 A C
ATOM 1381 O PRO A 189 20 .276 35 .975 4, .347 1 .00 20 .51 A O
ATOM 1382 N SER A 190 21 .285 34 .307 5, .492 1 .00 22 .89 A N
ATOM 1383 CA SER A 190 21 .033 34 .940 6, .796 1 .00 24 .65 A C
ATOM 1384 CB SER A 190 21 .685 34 .135 7 .932 1. .00 24 .76 A C
ATOM 1385 OG SER A 190 21 .082 32 .831 8. .046 1 .00 25 .85 A O
ATOM 1386 C SER A 190 19 .525 35 .098 7, .028 1. .00 25 .23 A C
ATOM 1387 O SER A 190 19 .080 35 .918 7, .850 1. .00 26 .47 A O
ATOM 1388 N PHE A 191 18, .723 34 .365 6, .258 1. .00 25 .36 A N
ATOM 1389 CA PHE A 191 17 .264 34 .446 6. .389 1 .00 25 .28 A C
ATOM 1390 CB PHE A 191 16, .643 33 .046 6, .156 1, .00 25 .00 A C
ATOM 1391 CG PHE A 191 16, .841 32 .089 7, .310 1. .00 23 .34 A C
ATOM 1392 CDl PHE A 191 17, .565 30 .932 7, .159 1, .00 21 .81 A C
ATOM 1393 CEl PHE A 191 17, .735 30 .052 8, .218 1, .00 22 .63 A C
ATOM 1394 CZ PHE A 191 17, .180 30 .341 9, .470 1, .00 21 .46 A C
ATOM 1395 CE2 PHE A 191 16, .449 31 .484 9, .631 1, .00 22 .61 A C
ATOM 1396 CD2 PHE A 191 16, .288 32 .361 8, .562 1, .00 25 .47 A C
ATOM 1397 C PHE A 191 16, .388 35 .561 5, .720 1, .00 25 .71 A C
ATOM 1398 O PHE A 191 15, .184 35 .500 5. .877 1, .00 27 .33 A O
ATOM 1399 N GLY A 192 16, .823 36 .552 4, .944 1, .00 26 .98 A N
ATOM 1400 CA GLY A 192 17, .639 36 .484 3, .783 1, .00 26 .29 A C
ATOM 1401 C GLY A 192 16, .795 36 .445 2, .478 1, .00 25 .37 A C
ATOM 1402 O GLY A 192 17, .008 35 .528 1, .733 1, ,00 25 .24 A O
ATOM 1403 N SER A 193 15, .858 37 .355 2, , 179 1, .00 24 .49 A N
ATOM 1404 CA SER A 193 15. .332 37 .444 0, .778 1, .00 24 .39 A C
ATOM 1405 CB SER A 193 14. .452 38 .689 0, .554 1, .00 24, .51 A C
ATOM 1406 OG SER A 193 13. ,058 38 .407 0, ,623 1, .00 25, .19 A O
ATOM 1407 C SER A 193 14, .664 36 .176 0, .133 1, .00 23 .94 A C
ATOM 1408 O SER A 193 14, .740 35 .973 1, ,085 1, .00 22 .27 A O
ATOM 1409 N TYR A 194 14, .037 35 .331 0. .949 1, .00 23, .39 A N
ATOM 1410 CA TYR A 194 13. .497 34 .046 0, .477 1, .00 23, .08 A C
ATOM 1411 CB TYR A 194 12. .407 33 .559 1. .439 1, .00 23, .87 A C
ATOM 1412 CG TYR A 194 11, .044 34. .129 1. .144 1, .00 27, .80 A C
ATOM 1413 CDl TYR A 194 10, .563 35 .240 1. .832 1, .00 31, .12 A C
ATOM 1414 CEl TYR A 194 9, .317 35, .775 1. .554 1, .00 32, .91 A C
ATOM 1415 CZ TYR A 194 8. ,525 35, .182 0. .591 1 , .00 34, .12 A C
ATOM 1416 OH TYR A 194 7, .282 35, .696 0. .311 1. .00 38, .22 A 0
ATOM 1417 CE2 TYR A 194 8. .974 34, .076 0. .108 1, .00 33, .28 A C
ATOM 1418 CD2 TYR A 194 10. .229 33, .556 0. .169 1. .00 31, .26 A c
ATOM 1419 C TYR A 194 14. .545 32, .930 0. ,289 1. .00 21, .59 A c
ATOM 1420 O TYR A 194 14. .225 31, ,848 0. .236 1. .00 20, .33 A 0
ATOM 1421 N ALA A 195 15. .785 33, .185 0. .695 1. .00 20, ,44 A N
ATOM 1422 CA ALA A 195 16. .838 32, .181 0. .610 1. .00 20, ,47 A C
ATOM 1423 CB ALA A 195 16. .915 31, .365 1. .892 1. .00 20, .41 A C
ATOM 1424 C ALA A 195 18. .222 32, .757 0. ,270 1. .00 19, .93 A C
ATOM 1425 O ALA A 195 19. .230 32 , .354 0. .877 1. .00 19, .21 A O
ATOM 1426 N ASP A 196 18. .264 33, .615 0. .750 1. .00 19, .15 A N
ATOM 1427 CA ASP A 196 19. .472 34, .355 1. .126 1 , .00 19, ,86 A C ATOM 1428 CB ASP A 196 19.264 35.861 0.919 1.00 20.04 A C
ATOM 1429 CG ASP A 196 18 .198 36 .453 1, .814 1 .00 22 .11 A C
ATOM 1430 ODl ASP A 196 18 .040 37 .693 1 .696 1 .00 23 .87 A O
ATOM 1431 OD2 ASP A 196 17 .461 35 .822 2 .649 1 .00 19 .55 A O
ATOM 1432 C ASP A 196 20 .025 34 .163 2, .549 1 .00 19 .90 A C
ATOM 1433 O ASP A 196 21 .092 34 .705 2, .869 1 .00 19 .61 A O
ATOM 1434 N ASN A 197 19 .326 33 .410 3, .394 1 .00 18 .85 A N
ATOM 1435 CA ASN A 197 19 .790 33 .177 4, .757 1 .00 18 .29 A C
ATOM 1436 CB ASN A 197 19 .410 34 .369 5 .644 1 .00 18 .68 A C
ATOM 1437 CG ASN A 197 20 .123 34 .360 7 .001 1 .00 19 .48 A C
ATOM 1438 ODl ASN A 197 20 .221 33 .319 7 .630 1 .00 16 .60 A O
ATOM 1439 ND2 ASN A 197 20 .603 35 .541 7, .455 1 .00 14 .55 A N
ATOM 1440 C ASN A 197 19 .198 31 .861 5, .304 1 .00 17 .30 A C
ATOM 1441 O ASN A 197 17 .986 31 .734 5, .463 1 .00 16 .96 A O
ATOM 1442 N ILE A 198 20 .066 30 .901 5, .608 1 .00 16 .67 A N
ATOM 1443 CA ILE A 198 19 .606 29 .557 5, .993 1 .00 16 .04 A C
ATOM 1444 CB ILE A 198 20 .771 28 .571 6, .020 1 .00 15 .64 A C
ATOM 1445 CGI ILE A 198 21 .724 28 .885 7, .179 1 .00 16 .27 A C
ATOM 1446 CDl ILE A 198 22 .734 27 .781 7, .490 1 .00 18 .08 A C
ATOM 1447 CG2 ILE A 198 21 .459 28 .530 4, .679 1 .00 16 .50 A C
ATOM 1448 C ILE A 198 18 .897 29 .560 7, .352 1 .00 15 .03 A C
ATOM 1449 O ILE A 198 18 .222 28 .605 7, .723 1. .00 15 .12 A O
ATOM 1450 N ASN A 199 19 .054 30 .642 8, .102 1 .00 14 .78 A N
ATOM 1451 CA ASN A 199 18 .316 30 .794 9, .344 1 .00 14 .68 A C
ATOM 1452 CB ASN A 199 19 .180 31 .609 10. .332 1 .00 14 .98 A C
ATOM 1453 CG ASN A 199 20 .487 30 .948 10. .708 1 .00 15 .69 A C
ATOM 1454 ODl ASN A 199 20 .560 29 .757 10. .907 1 .00 14 .98 A O
ATOM 1455 ND2 ASN A 199 21 .526 31 .768 10. .903 1 .00 20 .04 A N
ATOM 1456 C ASN A 199 17 .036 31 .597 9, .311 1 .00 14 .49 A C
ATOM 1457 O ASN A 199 16 .239 31 .490 10. .227 1 .00 14 .69 A O
ATOM 1458 N HIS A 200 16 .736 32 .328 8. ,241 1 .00 15 .73 A N
ATOM 1459 CA HIS A 200 15 .375 32 .648 7. .854 1, .00 14 .99 A C
ATOM 1460 CB HIS A 200 15 .338 33 .612 6. .641 1. .00 15 .17 A C
ATOM 1461 CG HIS A 200 16 .005 34 .942 6. ,871 1, .00 17 .24 A C
ATOM 1462 NDl HIS A 200 16 .242 35 .840 5. .842 1, .00 16 .94 A N
ATOM 1463 CEl HIS A 200 16 .842 36 .916 6. .327 1. .00 19 .73 A C
ATOM 1464 NE2 HIS A 200 17 .009 36 .751 7. .628 1, .00 17, .79 A N
ATOM 1465 CD2 HIS A 200 16 .469 35 .538 7. .999 1, ,00 18, .37 A C
ATOM 1466 C HIS A 200 14 .327 31 .581 7. .730 1, .00 15 .01 A C
ATOM 1467 O HIS A 200 14 .472 30 .673 6. .965 1, .00 14 , .41 A O
ATOM 1468 N VAL A 201 13 .251 31 .772 8. .496 1, .00 15, .72 A N
ATOM 1469 CA VAL A 201 12 .004 31 .059 8. .294 1, ,00 16, .37 A C
ATOM 1470 CB VAL A 201 11 .103 31 .185 9. .523 1, .00 16, .41 A C
ATOM 1471 CGI VAL A 201 9 .780 30, .428 9. ,297 1, .00 16, .12 A C
ATOM 1472 CG2 VAL A 201 11 .841 30, .668 10. .783 1, ,00 18, .01 A C
ATOM 1473 C VAL A 201 11 .313 31, .683 7. .089 1. .00 16, .94 A C
ATOM 1474 O VAL A 201 11 .250 32, .900 6. .973 1. ,00 17. .09 A O
ATOM 1475 N ALA A 202 10 .872 30, .865 6. ,143 1. ,00 17, ,70 A N
ATOM 1476 CA ALA A 202 10 .233 31, .396 4. ,949 1. ,00 17, .52 A C
ATOM 1477 CB ALA A 202 9 .859 30, .265 4. ,018 1. ,00 18, .76 A C
ATOM 1478 C ALA A 202 9 .000 32. .169 5. ,383 1. ,00 17. .84 A C
ATOM 1479 O ALA A 202 8 .263 31, ,734 6. ,263 1. ,00 16. .60 A O
ATOM 1480 N GLN A 203 8 .770 33. .332 4. ,783 1. ,00 18. .20 A N
ATOM 1481 CA GLN A 203 7, .629 34, .135 5. ,192 1. ,00 19. .45 A C
ATOM 1482 CB : BGLN A 203 7 .542 35, .347 4. ,260 0. ,40 19. .94 A C
ATOM 1483 CB , AGLN A 203 7, .529 35, .467 4. ,445 0. ,60 20. .39 A C
ATOM 1484 CG : BGLN A 203 7 .527 36, .681 4. ,943 0. ,40 22. .36 A C
ATOM 1485 CG . AGLN A 203 6 .748 36, .514 5. ,261 0. ,60 24. .58 A C
ATOM 1486 CD : BGLN A 203 6 .379 37, .556 4. ,452 0. ,40 25. ,52 A C
ATOM 1487 CD . AGLN A 203 7 .553 37, .090 6. ,439 0. ,60 28. ,39 A C
ATOM 1488 OE1BGLN A 203 5 .568 37, .122 3. 624 0. ,40 27. ,97 A O
ATOM 1489 OE1AGLN A 203 8 .525 37, .816 6. ,236 0. ,60 33. ,69 A O
ATOM 1490 NE2BGLN A 203 6 .299 38 , ,772 4. .972 0. ,40 24. ,61 A N ATOM 1491 NE2AGLN A 203 7.155 36.751 7.655 0.60 31.16 A N
ATOM 1492 C GLN A 203 6 .291 33 .391 5 .152 1 .00 18 .58 A C
ATOM 1493 O GLN A 203 5 .458 33 .580 6 .028 1 .00 18 .67 A O
ATOM 1494 N PHE A 204 6 .090 32 .533 4 .163 1 .00 17 .22 A N
ATOM 1495 CA PHE A 204 4 .805 31 .833 4 .027 1 .00 16 .75 A C
ATOM 1496 CB PHE A 204 4 .603 31 .335 2 .589 1 .00 16 .25 A C
ATOM 1497 CG PHE A 204 5 .720 30 .475 2 .093 1 .00 16 .52 A C
ATOM 1498 CDl PHE A 204 5 .857 29 .158 2 .526 1 .00 16 .66 A C
ATOM 1499 CEl PHE A 204 6 .893 28 .378 2 .083 1 .00 17 .13 A C
ATOM 1500 CZ PHE A 204 7 .854 28 .909 1, .234 1 .00 14 .67 A C
ATOM 1501 CE2 PHE A 204 7 .747 30 .231 0 .816 1 .00 16 .10 A C
ATOM 1502 CD2 PHE A 204 6 .693 31 .009 1 .267 1 .00 15 .39 A C
ATOM 1503 C PHE A 204 4 .670 30 .647 5, .018 1 .00 16 .21 A C
ATOM 1504 O PHE A 204 3 .570 30 .150 5, .198 1 .00 15 .42 A O
ATOM 1505 N SER A 205 5 .754 30 .223 5, .688 1 .00 15 .36 A N
ATOM 1506 CA SER A 205 5 .692 28 .983 6, .508 1 .00 15 .18 A C
ATOM 1507 CB SER A 205 7 .068 28 .579 7, .063 1 .00 15 .27 A C
ATOM 1508 OG SER A 205 7 .042 27 .254 7, .585 1 .00 15 .10 A O
ATOM 1509 C SER A 205 4 .657 29 .103 7, .615 1 .00 14 .84 A C
ATOM 1510 O SER A 205 4 .618 30 .092 8, .319 1 .00 16 .26 A O
ATOM 1511 N SER A 206 3 .764 28 .141 7, .753 1, .00 15 .82 A N
ATOM 1512 CA SER A 206 2. .751 28 .237 8, .818 1, .00 15 .73 A C
ATOM 1513 CB SER A 206 1 .714 27 .117 8, ,735 1, .00 16 .10 A C
ATOM 1514 OG SER A 206 0 .811 27, .350 7, .655 1, .00 14 .83 A O
ATOM 1515 C SER A 206 3. .421 28, .221 10. .186 1, .00 15 .92 A C
ATOM 1516 O SER A 206 4 .486 27, .589 10. ,362 1, ,00 15 .30 A O
ATOM 1517 N ARG A 207 2. .786 28, .928 11. .113 1, .00 15 .72 A N
ATOM 1518 CA ARG A 207 3. .289 29, .140 12. .455 1, .00 16 .34 A C
ATOM 1519 CB ARG A 207 3 .511 30, .636 12. .715 1, .00 16 .50 A C
ATOM 1520 CG ARG A 207 4 .189 31, .375 11. .542 1. .00 18 .52 A C
ATOM 1521 CD ARG A 207 5. .604 30, .916 11. .227 1, .00 20 .29 A C
ATOM 1522 NE ARG A 207 6, .146 31, .519 10. .012 1, .00 22 .13 A N
ATOM 1523 CZ ARG A 207 6, .821 32, .643 9. .981 1. .00 22. .20 A C
ATOM 1524 NHl ARG A 207 7, .056 33 , .294 11. .080 1. .00 25. .41 A N
ATOM 1525 NH2 ARG A 207 7, .256 33 , .124 8. ,838 1. ,00 24 .08 A N
ATOM 1526 C ARG A 207 2, .330 28, .556 13. ,471 1. .00 15 .97 A C
ATOM 1527 O ARG A 207 1, .096 28, .605 13. ,301 1. ,00 15. .01 A O
ATOM 1528 N GLY A 208 2, .903 27, .974 14. ,521 1. ,00 15, .16 A N
ATOM 1529 CA GLY A 208 2, .139 27 , .523 15. ,655 1. .00 16, .14 A C
ATOM 1530 C GLY A 208 1, .622 28, .688 16. ,476 1. .00 16, .50 A C
ATOM 1531 O GLY A 208 1, .753 29, .830 16. ,059 1. .00 17, .36 A O
ATOM 1532 N PRO A 209 0, .999 28, .423 17. ,617 1. ,00 17, .42 A N
ATOM 1533 CA PRO A 209 0, .727 27, .067 18. ,089 1. .00 17, .56 A C
ATOM 1534 CB PRO A 209 0, .407 27 , .269 19. ,579 1. .00 17, .58 A C
ATOM 1535 CG PRO A 209 -0, .088 28, .644 19. ,701 1. .00 18, .85 A C
ATOM 1536 CD PRO A 209 0, .477 29, .457 18. ,545 1. .00 17, .85 A C
ATOM 1537 C PRO A 209 -0, .483 26, .483 17. ,368 1. .00 17, .50 A C
ATOM 1538 O PRO A 209 -1, .094 27, .157 16. ,558 1. .00 18, .18 A O
ATOM 1539 N THR A 210 -0, .816 25, ,240 17. ,652 1. .00 16, .70 A N
ATOM 1540 CA THR A 210 -2, .050 24, .690 17. ,186 1. .00 17, .34 A C
ATOM 1541 CB THR A 210 -2, .042 23, .181 17. ,356 1. .00 16, .73 A C
ATOM 1542 OGl THR A 210 -1, .848 22, ,859 18. ,734 1. .00 18, .34 A O
ATOM 1543 CG2 THR A 210 -0, .833 22, .540 16. ,574 1. .00 16, .90 A C
ATOM 1544 C THR A 210 -3, .206 25, .327 17. ,987 1. .00 17, .55 A C
ATOM 1545 O THR A 210 -2, .990 26. .095 18. ,930 1. .00 16, .40 A O
ATOM 1546 N ARG A 211 -4, .421 24. .979 17. ,623 1. .00 18, .89 A N
ATOM 1547 CA ARG A 211 -5, ,595 25. .577 18. ,264 1. .00 20, .56 A C
ATOM 1548 CB ARG A 211 -6, .884 25, .056 17. ,638 1. .00 21, .16 A C
ATOM 1549 CG ARG A 211 -8, .149 25. .719 18. ,255 1. .00 25, ,92 A C
ATOM 1550 CD ARG A 211 -9, .325 25. .804 17. ,301 1. .00 31, ,08 A C
ATOM 1551 NE ARG A 211 -8, .956 26. .457 16. ,042 1. ,00 35, .63 A N
ATOM 1552 CZ ARG A 211 -9, ,626 26. ,296 14. ,905 1. ,00 38, .91 A C
ATOM 1553 NHl ARG A 211 -10, .707 25. .516 14. ,876 1. .00 40, .64 A N ATOM 1554 NH2 ARG A 211 -9.225 26.911 13..795 1.00 37.74 A N
ATOM 1555 C ARG A 211 -5. .591 25 .308 19. .768 1 .00 20 .16 A C
ATOM 1556 O ARG A 211 -5. .983 26 .180 20. .539 1 .00 19 .93 A O
ATOM 1557 N ASP A 212 -5 .120 24 .121 20. .185 1 .00 19 .40 A N
ATOM 1558 CA ASP A 212 -5 .031 23 .791 21, .616 1 .00 18 .64 A C
ATOM 1559 CB ASP A 212 -5 .346 22 .306 21, .877 1 .00 18 .58 A C
ATOM 1560 CG ASP A 212 -4 .318 21 .356 21. .254 1 .00 16 .59 A C
ATOM 1561 ODl ASP A 212 -4 .255 20 .180 21. .679 1 .00 16 .40 A O
ATOM 1562 OD2 ASP A 212 -3 .545 21 .688 20, .339 1 .00 17 .56 A O
ATOM 1563 C ASP A 212 -3 .693 24 .160 22, .255 1 .00 18 .83 A C
ATOM 1564 O ASP A 212 -3 .387 23 .707 23, .370 1 .00 19 .22 A O
ATOM 1565 N GLY A 213 -2 .902 24 .966 21, .556 1 .00 18 .29 A N
ATOM 1566 CA GLY A 213 -1 .698 25 .572 22, .111 1 .00 18 .10 A C
ATOM 1567 C GLY A 213 -0. .439 24 .713 22, .065 1 .00 17 .75 A C
ATOM 1568 O GLY A 213 0. .517 24 .998 22, ,785 1 .00 18 .06 A O
ATOM 1569 N ARG A 214 -0 .431 23 .665 21. .242 1 .00 16 .43 A N
ATOM 1570 CA ARG A 214 0 .757 22 .826 21, .110 1 .00 16 .72 A C
ATOM 1571 CB ARG A 214 0. .403 21 .461 20, .536 1 .00 16 .17 A C
ATOM 1572 CG ARG A 214 -0 .276 20 .553 21, .473 1 .00 16 .40 A C
ATOM 1573 CD ARG A 214 -0 .753 19 .301 20. .814 1 .00 16 .61 A C
ATOM 1574 NE ARG A 214 -1 .771 19 .613 19. .826 1 .00 16 .67 A N
ATOM 1575 CZ ARG A 214 -1, .740 19 .297 18. .531 1 .00 16 .71 A C
ATOM 1576 NHl ARG A 214 -0, .720 18 .628 17. .981 1 .00 16 .55 A N
ATOM 1577 NH2 ARG A 214 -2, .762 19 .664 17. .776 1 .00 14, .42 A N
ATOM 1578 C ARG A 214 1, .772 23 .493 20. ,203 1 .00 16 .16 A C
ATOM 1579 O ARG A 214 1, .403 24 .306 19. ,344 1 .00 16 .86 A O
ATOM 1580 N ILE A 215 3 .046 23 .168 20. .396 1 .00 15 .82 A N
ATOM 1581 CA ILE A 215 4 .107 23 .640 19. .516 1 .00 15 .40 A C
ATOM 1582 CB ILE A 215 5 .503 23 .498 20. ,175 1 .00 16 .18 A C
ATOM 1583 CGI ILE A 215 5 .600 24 .351 21, .454 1 .00 17 .35 A C
ATOM 1584 CDl ILE A 215 5 .526 25 .842 21, .181 1 .00 20 .01 A C
ATOM 1585 CG2 ILE A 215 6, ,606 23 .898 19. .191 1 .00 15 .87 A C
ATOM 1586 C ILE A 215 4, .100 22 .834 18. .214 1 .00 15 .40 A C
ATOM 1587 O ILE A 215 4, .316 21 .616 18. .227 1 .00 14 .96 A O
ATOM 1588 N LYS A 216 3, .841 23 .536 17. .117 1 .00 14 .61 A N
ATOM 1589 CA LYS A 216 4, .072 23 .062 15. .745 1, .00 14 .64 A C
ATOM 1590 CB LYS A 216 2, .765 22 .616 15. ,067 1, .00 13, .98 A C
ATOM 1591 CG LYS A 216 2, .190 21 .271 15. ,526 1, .00 13, .46 A C
ATOM 1592 CD LYS A 216 3, .073 20 .102 15. ,117 1, .00 14, .36 A C
ATOM 1593 CE LYS A 216 2, .427 18 .754 15. .453 1, .00 13, .24 A C
ATOM 1594 NZ LYS A 216 3, ,042 17 .577 14. .739 1, .00 8, .08 A N
ATOM 1595 C LYS A 216 4, ,632 24 .269 14. .984 1, .00 14, .70 A C
ATOM 1596 O LYS A 216 4. .336 25 .428 15. .358 1, .00 13, ,92 A O
ATOM 1597 N PRO A 217 5, .410 24 .032 13. ,921 1, ,00 14 , .59 A N
ATOM 1598 CA PRO A 217 5. .788 22, .691 13. ,468 1, .00 13 , .65 A C
ATOM 1599 CB PRO A 217 6. ,452 22, .944 12. ,115 1, .00 14, ,58 A C
ATOM 1600 CG PRO A 217 6. ,934 24, .356 12. ,178 1, .00 15, .23 A C
ATOM 1601 CD PRO A 217 6. ,012 25, .077 13. 086 1. .00 14. ,44 A C
ATOM 1602 C PRO A 217 6. .818 22, .089 14. ,401 1. .00 12 , .99 A C
ATOM 1603 O PRO A 217 7, ,262 22 .738 15. ,379 1, .00 12. .11 A O
ATOM 1604 N ASP A 218 7, .201 20 .847 14. , 126 1, .00 11. .74 A N
ATOM 1605 CA ASP A 218 8, .188 20 .214 14. ,974 1, .00 11, .35 A C
ATOM 1606 CB ASP A 218 8, .033 18 .694 14. .962 1, .00 11, .47 A C
ATOM 1607 CG ASP A 218 6, .672 18 .241 15. ,451 1, .00 11. .82 A C
ATOM 1608 ODl ASP A 218 6, .440 18 .370 16. ,680 1, .00 10, .57 A O
ATOM 1609 OD2 ASP A 218 5, .810 17 .726 14. ,671 1, .00 11, .50 A O
ATOM 1610 C ASP A 218 9, .619 20 .566 14. ,610 1, .00 11, ,30 A C
ATOM 1611 O ASP A 218 10. .441 20 .772 15. ,501 1, .00 10, .85 A O
ATOM 1612 N VAL A 219 9. .928 20 .516 13. ,314 1, .00 11, .61 A N
ATOM 1613 CA VAL A 219 11. .254 20, .829 12. ,815 1, .00 12, .37 A C
ATOM 1614 CB VAL A 219 12. .118 19 .589 12. ,602 1, .00 12 , .19 A C
ATOM 1615 CGI VAL A 219 12. .401 18 .867 13. .933 1, .00 14, .24 A C
ATOM 1616 CG2 VAL A 219 11. .485 18 .660 11. ,587 1, .00 13. .54 A C ATOM 1617 C VAL A 219 11,,148 21.568 11.471 1.00 12.33 A C
ATOM 1618 O VAL A 219 10, .083 21 .624 10 .851 1 .00 12 .34 A O
ATOM 1619 N MET A 220 12, .266 22 .139 11 .057 1 .00 11 .78 A N
ATOM 1620 CA MET A 220 12, .365 22 .930 9 .852 1 .00 11 .89 A C
ATOM 1621 CB MET A 220 12 .798 24 .371 10 .167 1 .00 11 .30 A C
ATOM 1622 CG MET A 220 12, .025 25 .058 11 .255 1 .00 11 .64 A C
ATOM 1623 SD MET A 220 10 .310 25 .322 10 .860 1 .00 12 .02 A S
ATOM 1624 CE MET A 220 10 .416 26 .727 9 .791 1 .00 11 .35 A C
ATOM 1625 C MET A 220 13 .398 22 .343 8 .902 1 .00 12 .21 A C
ATOM 1626 O MET A 220 14 .368 21 .731 9 .321 1 .00 12 .24 A O
ATOM 1627 N ALA A 221 13 .175 22 .556 7 .613 1 .00 12 .60 A N
ATOM 1628 CA ALA A 221 14 .198 22 .324 6 .605 1 .00 12 .90 A C
ATOM 1629 CB ALA A 221 14 .098 20 .912 6 .081 1 .00 12 .23 A C
ATOM 1630 C ALA A 221 14 .064 23 .341 5 .464 1 .00 13 .77 A C
ATOM 1631 O ALA A 221 13 .029 24 .027 5 .312 1 .00 14 .46 A O
ATOM 1632 N PRO A 222 15, .116 23, .487 4, .687 1 .00 13 .99 A N
ATOM 1633 CA PRO A 222 15, .059 24, .393 3, .543 1 .00 14 .73 A C
ATOM 1634 CB PRO A 222 16, .387 24, .159 2, .845 1 .00 13 .69 A C
ATOM 1635 CG PRO A 222 17, ,290 23, .676 3. .892 1 .00 15 .06 A C
ATOM 1636 CD PRO A 222 16, .433 22 .855 4, .830 1 .00 14 .39 A C
ATOM 1637 C PRO A 222 13, .896 24, .044 2, .622 1 .00 14 .82 A C
ATOM 1638 O PRO A 222 13, ,719 22, .847 2, .284 1 .00 15 .21 A O
ATOM 1639 N GLY A 223 13, .178 25, ,069 2, .193 1 .00 14 .57 A N
ATOM 1640 CA GLY A 223 11, .996 24, .910 1, ,373 1 .00 15 .06 A C
ATOM 1641 C GLY A 223 11, ,779 26, .046 0, .383 1, .00 14, .81 A C
ATOM 1642 O GLY A 223 10, .661 26, .268 -0. .039 1 .00 15, .67 A O
ATOM 1643 N THR A 224 12, .822 26, .799 0, .049 1, .00 13, .98 A N
ATOM 1644 CA THR A 224 12, .706 27, .772 -1, .007 1, .00 14, .01 A C
ATOM 1645 CB THR A 224 12, .912 29 .229 -0, .517 1, .00 13, .98 A C
ATOM 1646 OGl THR A 224 14, .220 29, .350 0, .047 1, .00 13. .39 A O
ATOM 1647 CG2 THR A 224 11, .952 29, .585 0. ,597 1, .00 14. .59 A C
ATOM 1648 C THR A 224 13, .729 27, .449 -2, .072 1, .00 14, ,02 A C
ATOM 1649 O THR A 224 14, .813 26, .932 -1, ,791 1, .00 14, .13 A O
ATOM 1650 N TYR A 225 13, .389 27, .786 -3, .308 1, .00 14, .73 A N
ATOM 1651 CA TYR A 225 14, .270 27 .528 -4, .441 1, .00 14, .78 A C
ATOM 1652 CB TYR A 225 15, .197 28 .726 -4, .686 1, .00 15, .26 A C
ATOM 1653 CG TYR A 225 14, .502 29 .848 -5, ,398 1, .00 15, .90 A C
ATOM 1654 CDl TYR A 225 14, .027 30 .940 -4, ,692 1, ,00 18. .12 A C
ATOM 1655 CEl TYR A 225 13, .349 31 .960 -5, .301 1, .00 18, ,57 A C
ATOM 1656 CZ TYR A 225 13 .100 31 .918 -6, .659 1, .00 19. .46 A C
ATOM 1657 OH TYR A 225 12 .391 32 .974 -7, .207 1, .00 20. .70 A O
ATOM 1658 CE2 TYR A 225 13 .510 30 .844 -7, .404 1, .00 18, ,30 A C
ATOM 1659 CD2 TYR A 225 14 .225 29 .788 -6, .771 1, .00 19. .15 A C
ATOM 1660 C TYR A 225 15 .022 26 .196 -4, .331 1, .00 14. .79 A C
ATOM 1661 O TYR A 225 16 .252 26 .119 -4, .395 1, .00 15. .52 A O
ATOM 1662 N ILE A 226 14 .248 25 .130 -4, ,186 1, .00 15. .07 A N
ATOM 1663 CA ILE A 226 14 .773 23 .759 -4, .155 1, ,00 14. .45 A C
ATOM 1664 CB ILE A 226 13 .904 22 .866 -3, .254 1, .00 14. .35 A C
ATOM 1665 CGI ILE A 226 13 .906 23 .341 -1, .789 1, .00 15. .47 A C
ATOM 1666 CDl ILE A 226 15 .239 23 .250 -1, .085 1, ,00 16. .51 A C
ATOM 1667 CG2 ILE A 226 14 .312 21 .400 -3, .377 1, .00 14. .04 A C
ATOM 1668 C ILE A 226 14 .780 23 .205 -5, ,580 1. .00 14. .24 A C
ATOM 1669 O ILE A 226 13 .778 23 .188 -6, .245 1, .00 13. .78 A O
ATOM 1670 N LEU A 227 15 .937 22 .753 -6, .022 1, .00 14. ,94 A N
ATOM 1671 CA LEU A 227 16 .141 22 .230 -7, ,359 1, .00 14. .97 A C
ATOM 1672 CB LEU A 227 17 .541 22 .653 -7, .827 1, .00 15. .61 A C
ATOM 1673 CG LEU A 227 17 .950 22 .137 -9, .196 1. .00 16. .60 A C
ATOM 1674 CDl LEU A 227 16 .899 22 .508 -10. .231 1. .00 16. ,90 A C
ATOM 1675 CD2 LEU A 227 19 .340 22 .669 -9, .559 1, .00 18. ,98 A C
ATOM 1676 C LEU A 227 16 .010 20 .708 -7. ,284 1. .00 14. ,76 A C
ATOM 1677 O LEU A 227 16 .803 20 .038 -6. .602 1. .00 15. .34 A O
ATOM 1678 N SER A 228 14 .970 20, .179 -7. ,924 1. ,00 14. 03 A N
ATOM 1679 CA SER A 228 14 .665 18, .752 -7. ,871 1. ,00 14. 07 A C ATOM 1680 CB SER A 228 13.701 18.448 -6.701 1.00 13.81 A C
ATOM 1681 OG SER A 228 13 .631 17 .038 -6 .453 1 .00 12 .38 A O
ATOM 1682 C SER A 228 14 .061 18 .319 -9 .208 1 .00 14 .98 A C
ATOM 1683 O SER A 228 13 .971 19 .115 -10 .133 1 .00 15 .43 A O
ATOM 1684 N ALA A 229 13 .626 17 .067 -9 .278 1 .00 14 .57 A N
ATOM 1685 CA ALA A 229 13 .155 16 .454 -10 .516 1 .00 14 .87 A C
ATOM 1686 CB ALA A 229 12 .824 14 .945 -10 .268 1 .00 14 .78 A C
ATOM 1687 C ALA A 229 11 .939 17 .135 -11 .086 1 .00 14 .83 A C
ATOM 1688 O ALA A 229 10 .939 17 .411 -10 .376 1 .00 14 .22 A O
ATOM 1689 N ARG A 230 12 .027 17 .381 -12 .394 1 .00 14 .42 A N
ATOM 1690 CA ARG A 230 10 .974 18 .013 -13 .155 1 .00 14 .49 A C
ATOM 1691 CB ARG A 230 11 .553 19 .008 -14 .137 1 .00 14 .60 A C
ATOM 1692 CG ARG A 230 10 .516 19 .626 -15 .065 1 .00 16 .55 A C
ATOM 1693 CD ARG A 230 11 .044 20 .792 -15 .934 1 .00 19 .98 A C
ATOM 1694 NE ARG A 230 9 .940 21 .308 -16 .751 1 .00 19 .63 A N
ATOM 1695 CZ ARG A 230 9 .692 22 .581 -16 .995 1 .00 21 .34 A C
ATOM 1696 NHl ARG A 230 10 .502 23 .547 -16 .545 1 .00 21 .55 A N
ATOM 1697 NH2 ARG A 230 8 .617 22 .898 -17 .730 1 .00 20 .71 A N
ATOM 1698 C ARG A 230 10 .232 16 .947 -13 ,948 1 .00 14 .81 A C
ATOM 1699 O ARG A 230 10 .838 16 .237 -14 .762 1 .00 14 .40 A O
ATOM 1700 N SER A 231 8 .931 16 .837 -13. .703 1 .00 14 .68 A N
ATOM 1701 CA SER A 231 8 .106 15 .937 -14. .463 1 .00 15 .32 A C
ATOM 1702 CB SER A 231 6 .660 16 .034 -14, .030 1 .00 15 .75 A C
ATOM 1703 OG SER A 231 5 .836 15 .317 -14, .947 1 .00 16 .08 A O
ATOM 1704 C SER A 231 8 .176 16 .325 -15, .956 1 .00 15 .44 A C
ATOM 1705 O SER A 231 8 .087 17 .494 -16, .306 1 .00 13 .31 A O
ATOM 1706 N SER A 232 8 .295 15 .321 -16, .802 1. .00 15 .69 A N
ATOM 1707 CA SER A 232 8 .323 15 .494 -18, .255 1 .00 16 .52 A C
ATOM 1708 CB SER A 232 8 .682 14 .156 -18, .906 1, .00 16 .29 A C
ATOM 1709 OG SER A 232 7 .610 13 .191 -18, .730 1, .00 16 .72 A O
ATOM 1710 C SER A 232 7, .004 16 .050 -18, .820 1, .00 18 .10 A C
ATOM 1711 O SER A 232 6, .970 16 .540 -19, ,945 1, .00 18 .08 A O
ATOM 1712 N LEU A 233 5, .924 16 .005 -18. .040 1, .00 18 .99 A N
ATOM 1713 CA LEU A 233 4, .647 16, .550 -18, ,466 1, .00 19 .87 A C
ATOM 1714 CB LEU A 233 3. .503 15 .655 -17, .989 1, .00 20 .52 A C
ATOM 1715 CG LEU A 233 3, .579 14 .202 -18, .428 1, .00 22 .45 A C
ATOM 1716 CDl LEU A 233 2, .344 13 .472 -17, ,943 1, .00 25 .84 A C
ATOM 1717 CD2 LEU A 233 3, .683 14 .146 -19, ,948 1, .00 26 .24 A C
ATOM 1718 C LEU A 233 4 , .357 17 .956 -17, .940 1, .00 20 .22 A C
ATOM 1719 O LEU A 233 3, .365 18 .546 -18. .345 1, .00 20 .30 A O
ATOM 1720 N ALA A 234 5, .164 18 .485 -17. .016 1, .00 18 .84 A N
ATOM 1721 CA ALA A 234 4, .768 19, .731 -16. .365 1. .00 19 .34 A C
ATOM 1722 CB ALA A 234 5, .297 19 .781 -14. .958 1, .00 18 .58 A C
ATOM 1723 C ALA A 234 5, .197 20, .991 -17. .153 1, .00 19 .93 A C
ATOM 1724 O ALA A 234 6, .300 21, .037 -17. .701 1, .00 20 .41 A O
ATOM 1725 N PRO A 235 4, .325 21, .989 -17. .197 1, ,00 20, .75 A N
ATOM 1726 CA PRO A 235 4, .642 23, .288 -17. ,802 1, .00 21 .88 A C
ATOM 1727 CB PRO A 235 3, .271 23. .921 -17. .981 1, .00 21, .83 A C
ATOM 1728 CG PRO A 235 2, .429 23, .326 -16. .902 1, ,00 21, .93 A C
ATOM 1729 CD PRO A 235 2 , .947 21, .944 -16. .677 1, .00 21, .22 A C
ATOM 1730 C PRO A 235 5. .495 24, .199 -16. .885 1, .00 22, .70 A C
ATOM 1731 O PRO A 235 5. .513 23, .970 -15. .671 1, .00 21, .06 A O
ATOM 1732 N ASP A 236 6. .150 25, .204 -17. .489 1, .00 24, .00 A N
ATOM 1733 CA ASP A 236 6. .960 26, .228 -16. .795 1. .00 24 , ,41 A C
ATOM 1734 CB ASP A 236 7. .455 27, .332 -17. ,750 1, .00 24, .21 A C
ATOM 1735 CG ASP A 236 8. .603 26, .838 -18. .636 1, .00 25, ,42 A C
ATOM 1736 ODl ASP A 236 9. .214 27, ,656 -19. .365 1. .00 25, .18 A O
ATOM 1737 OD2 ASP A 236 8. .990 25, .634 -18. ,674 1, .00 23, .87 A O
ATOM 1738 C ASP A 236 6. .263 26, .644 -15. .520 1 , .00 24 , .33 A C
ATOM 1739 O ASP A 236 6. .919 27, ,035 -14. .558 1, ,00 24, ,16 A O
ATOM 1740 N SER A 237 4. .933 26. .677 -15. .491 1. .00 25, .32 A N
ATOM 1741 CA SER A 237 4. .179 27. .723 -14. .873 1. .00 24, .89 A C
ATOM 1742 CB SER A 237 2, .801 27. .926 -15. .490 1, .00 25, .98 A C ATOM 1743 OG SER A 237 2.035 26.723 -15.436 1.00 27.95 A O
ATOM 1744 C SER A 237 4 .027 26 .960 -13 .487 1 .00 24 .14 A C
ATOM 1745 O SER A 237 3 .588 27 .516 -12 .495 1 .00 23 .01 A O
ATOM 1746 N SER A 238 4 .363 25 .660 -13, .448 1 .00 22 .59 A N
ATOM 1747 CA SER A 238 4 .313 24 .861 -12, .201 1 .00 22 .41 A C
ATOM 1748 CB SER A 238 4 .238 23 .344 -12, .501 1 .00 22 .11 A C
ATOM 1749 OG SER A 238 3 .046 22 .968 -13, .146 1 .00 22 .38 A O
ATOM 1750 C SER A 238 5 .543 25 .045 -11 .295 1 .00 21 .86 A C
ATOM 1751 O SER A 238 5 .550 24 .542 -10 .184 1 .00 22 .29 A O
ATOM 1752 N PHE A 239 6 .568 25 .744 -11 .789 1 .00 21 .32 A N
ATOM 1753 CA PHE A 239 7 .847 25 .899 -11, .108 1 .00 20 .67 A C
ATOM 1754 CB PHE A 239 8 .966 25 .299 -11 .966 1 .00 20 .17 A C
ATOM 1755 CG PHE A 239 8 .736 23 .854 -12 .294 1 .00 20 .18 A C
ATOM 1756 CDl PHE A 239 8 .964 22 .881 -11, .344 1 .00 18 .34 A C
ATOM 1757 CEl PHE A 239 8 .686 21 .573 -11 .600 1 .00 15 .67 A C
ATOM 1758 CZ PHE A 239 8 .194 21 .184 -12 .814 1 .00 16 .81 A C
ATOM 1759 CE2 PHE A 239 7 .924 22 .128 -13, .775 1 .00 16 .81 A C
ATOM 1760 CD2 PHE A 239 8 .194 23 .466 -13 .520 1 .00 18 .63 A C
ATOM 1761 C PHE A 239 8 .124 27 .370 -10, .775 1 .00 20 .81 A C
ATOM 1762 O PHE A 239 7 .589 28 .283 -11. .404 1 .00 19 .94 A O
ATOM 1763 N TRP A 240 8 .927 27 .575 -9. .743 1 .00 20 .46 A N
ATOM 1764 CA TRP A 240 9 .420 28 .913 -9, .382 1 .00 21 .13 A C
ATOM 1765 CB TRP A 240 10 .192 28 .842 -8 .055 1 .00 21 .05 A C
ATOM 1766 CG TRP A 240 9 .324 28 .850 -6, .857 1 .00 22 .76 A C
ATOM 1767 CDl TRP A 240 8 .027 28 .446 -6 .782 1 .00 23 .97 A C
ATOM 1768 NE1 TRP A 240 7 .548 28 .624 -5 .509 1 .00 24 .62 A N
ATOM 1769 CE2 TRP A 240 8. .547 29 .148 -4, .726 1, .00 24 , .31 A C
ATOM 1770 CD2 TRP A 240 9, .677 29 .302 -5, .537 1, .00 23 .24 A C
ATOM 1771 CE3 TRP A 240 10, .839 29 .811 -4, .966 1, .00 24, .95 A C
ATOM 1772 CZ3 TRP A 240 10, .833 30, .146 -3. .637 1, .00 24 , .14 A C
ATOM 1773 CH2 TRP A 240 9, .682 29, .991 -2. .857 1, .00 23, .89 A C
ATOM 1774 CZ2 TRP A 240 8, .542 29, .483 -3, ,378 1, .00 25, .05 A C
ATOM 1775 C TRP A 240 10, .355 29 .466 -10. .461 1, .00 20, ,95 A C
ATOM 1776 O TRP A 240 10, .419 30 .673 -10. .703 1, .00 20, .42 A O
ATOM 1777 N ALA A 241 11, .097 28 .566 -11, .080 1, .00 21, .11 A N
ATOM 1778 CA ALA A 241 12, .022 28 .907 -12, .149 1, .00 21, .52 A C
ATOM 1779 CB ALA A 241 13, .243 29 .629 -11, .606 1, .00 21, .93 A C
ATOM 1780 C ALA A 241 12, .466 27 .641 -12, .801 1 .00 21, .79 A C
ATOM 1781 O ALA A 241 12, .440 26 .569 -12. ,169 1 .00 22 .05 A O
ATOM 1782 N ASN A 242 12, .929 27 .769 -14. ,040 1, .00 22 .09 A N
ATOM 1783 CA ASN A 242 13, .481 26 .656 -14, ,800 1 .00 22 .74 A C
ATOM 1784 CB ASN A 242 13, .397 26 .962 -16, .322 1 .00 22 .69 A C
ATOM 1785 CG ASN A 242 11. .960 27 .071 -16. .828 1 .00 22 .96 A C
ATOM 1786 ODl ASN A 242 11, .024 26 .578 -16. , 198 1 .00 21 .33 A O
ATOM 1787 ND2 ASN A 242 11, .782 27 .727 -17. .969 1 .00 21 .31 A N
ATOM 1788 C ASN A 242 14, .927 26 .359 -14. .458 1 .00 23 .18 A C
ATOM 1789 O ASN A 242 15 .634 27 .194 -13, .902 1 .00 23 .35 A O
ATOM 1790 N HIS A 243 15 .375 25 .169 -14, .820 1 .00 24 .21 A N
ATOM 1791 CA HIS A 243 16 .802 24 .875 -14, .862 1 .00 25 .06 A C
ATOM 1792 CB HIS A 243 17, .234 24 , .062 -13. .653 1, .00 25. ,31 A C
ATOM 1793 CG HIS A 243 18, .703 23, .809 -13. .595 1, .00 27. .16 A C
ATOM 1794 NDl HIS A 243 19, .599 24, .733 -13. .086 1, .00 30. .11 A N
ATOM 1795 CEl HIS A 243 20, .820 24 , .231 -13. .152 1, .00 30, .29 A C
ATOM 1796 NE2 HIS A 243 20, .752 23. .036 -13. .713 1, .00 29, .63 A N
ATOM 1797 CD2 HIS A 243 19, .442 22, .754 -14. .008 1, ,00 28, .73 A C
ATOM 1798 C HIS A 243 17, .158 24 , .144 -16. .162 1. .00 26, .02 A C
ATOM 1799 O HIS A 243 17, .851 24 .726 -17. .003 1. .00 25, .77 A O
ATOM 1800 N ASP A 244 16, .711 22 .880 -16, .299 1, .00 26, .20 A N
ATOM 1801 CA ASP A 244 16, ,757 22 .137 -17, .584 1, .00 27, .35 A C
ATOM 1802 CB ASP A 244 17, .972 21 .252 -17, .646 1, .00 28, .02 A C
ATOM 1803 CG ASP A 244 18, .211 20 .272 -16, .546 1, .00 29 .28 A C
ATOM 1804 ODl ASP A 244 19, .393 20 .188 -16, .099 1, .00 34 .73 A O
ATOM 1805 OD2 ASP A 244 17, .310 19 .568 -16, , 056 1, .00 28 .43 A O ATOM 1806 C ASP A 244 15,.427 21.429 -17,.760 1.00 27.57 A C
ATOM 1807 O ASP A 244 14, .751 21 .208 -16, .721 1 .00 27 .11 A O
ATOM 1808 N SER A 245 15, .290 20 .734 -18, ,836 1 .00 27 .83 A N
ATOM 1809 CA SER A 245 14 .559 19 .557 -19 .209 1 .00 26 .73 A C
ATOM 1810 CB SER A 245 15 .083 18 .972 -20 .483 1 .00 26 .99 A C
ATOM 1811 OG SER A 245 15 .792 17 .785 -20 .481 1 .00 27 .12 A O
ATOM 1812 C SER A 245 14 .234 18 .594 -18 .102 1 .00 25 .28 A C
ATOM 1813 O SER A 245 13 .146 17 .973 -18 .152 1 .00 24 .66 A O
ATOM 1814 N LYS A 246 15 .122 18 .339 -17 .176 1 .00 23 .51 A N
ATOM 1815 CA LYS A 246 14 .918 17 .304 -16 .177 1 .00 22 .90 A C
ATOM 1816 CB LYS A 246 15 .977 16 .205 -16, .332 1 .00 23 .64 A C
ATOM 1817 CG LYS A 246 15 .852 15 .384 -17, .600 1 .00 26 .42 A C
ATOM 1818 CD LYS A 246 17 .094 14 .548 -17, .859 1 .00 29 .39 A C
ATOM 1819 CE LYS A 246 16, .880 13 .584 -19, .018 1 .00 32 .94 A C
ATOM 1820 NZ LYS A 246 18 .070 13 .501 -19, .908 1 .00 37 .30 A N
ATOM 1821 C LYS A 246 14 .812 17 .762 -14, .740 1 .00 21 .77 A C
ATOM 1822 O LYS A 246 14, .396 17 .059 -13, .828 1 .00 19 .24 A O
ATOM 1823 N TYR A 247 15 .126 19 .026 -14, .452 1 .00 20 .43 A N
ATOM 1824 CA TYR A 247 15, .144 19 .544 -13, .079 1 .00 19 .83 A C
ATOM 1825 CB TYR A 247 16, .541 19 .398 -12, ,456 1 .00 19 .35 A C
ATOM 1826 CG TYR A 247 17, .007 17 .966 -12, .434 1 .00 19 .14 A C
ATOM 1827 CDl TYR A 247 17, .784 17 .442 -13, .482 1, .00 21 .08 A C
ATOM 1828 CEl TYR A 247 18, ,170 16 .121 -13, .489 1, .00 17 .97 A C
ATOM 1829 CZ TYR A 247 17, .780 15 .292 -12. .458 1, .00 19 .97 A C
ATOM 1830 OH TYR A 247 18. .159 13 .964 -12. .465 1, .00 18 .06 A O
ATOM 1831 CE2 TYR A 247 16, .999 15 .781 -11, .417 1, .00 18 .19 A C
ATOM 1832 CD2 TYR A 247 16 .630 17 .109 -11, .408 1 .00 19 .09 A C
ATOM 1833 C TYR A 247 14 .697 21 .003 -13. .069 1 .00 18 .89 A C
ATOM 1834 O TYR A 247 15 .017 21 .761 -13. .994 1 .00 18 .71 A O
ATOM 1835 N ALA A 248 13 .936 21 .385 -12. .046 1, .00 17 .07 A N
ATOM 1836 CA ALA A 248 13 .512 22 .769 -11. ,893 1 .00 16 .28 A C
ATOM 1837 CB ALA A 248 12 .294 23 .035 -12, .733 1, .00 15 .85 A C
ATOM 1838 C ALA A 248 13 .253 23 .110 -10, .425 1, .00 15 .75 A C
ATOM 1839 O ALA A 248 13 .358 22 .236 -9, .549 1, .00 15 .81 A O
ATOM 1840 N TYR A 249 12 .956 24 .384 -10, .174 1, .00 15 .33 A N
ATOM 1841 CA TYR A 249 12 .910 24 .949 -8, .832 1, .00 15 .08 A C
ATOM 1842 CB TYR A 249 13 .520 26 .336 -8, .802 1, .00 15, .54 A C
ATOM 1843 CG TYR A 249 14, .999 26 .398 -9. .087 1, ,00 15, .33 A C
ATOM 1844 CDl TYR A 249 15, .470 26 .675 -10, .370 1. .00 17, .29 A C
ATOM 1845 CEl TYR A 249 16, .829 26 .754 -10. ,640 1, .00 16, .19 A C
ATOM 1846 CZ TYR A 249 17, .741 26 .557 -9. .608 1, .00 18, .72 A C
ATOM 1847 OH TYR A 249 19. .088 26 .649 -9. .839 1. ,00 21, .92 A O
ATOM 1848 CE2 TYR A 249 17, .306 26 .287 -8. .330 1. .00 18, .20 A C
ATOM 1849 CD2 TYR A 249 15, .930 26, .207 -8. .070 1. .00 17, .03 A C
ATOM 1850 C TYR A 249 11, .497 25, .078 -8. ,358 1. ,00 15, .50 A C
ATOM 1851 O TYR A 249 10, ,599 25, .480 -9. ,122 1. .00 16. .06 A 0
ATOM 1852 N MET A 250 11. .291 24, .749 -7. ,082 1. ,00 15. .10 A N
ATOM 1853 CA MET A 250 10. .015 24, .967 -6. ,430 1. ,00 15. ,40 A C
ATOM 1854 CB MET A 250 9, .153 23, .703 -6. ,542 1. ,00 15. ,90 A C
ATOM 1855 CG MET A 250 7 .677 23 .947 -6. .729 1. ,00 19, .64 A C
ATOM 1856 SD MET A 250 6. .677 22 .370 -6. .869 1. ,00 23, .44 A S
ATOM 1857 CE MET A 250 7. .321 21, .709 -8. .163 1. .00 22. .96 A C
ATOM 1858 C MET A 250 10, ,274 25, .318 -4. ,966 1. ,00 15. .09 A C
ATOM 1859 O MET A 250 11, .366 25, .081 -4. ,440 1. ,00 15. .78 A O
ATOM 1860 N GLY A 251 9, .279 25, .888 -4. ,314 1. ,00 14. ,69 A N
ATOM 1861 CA GLY A 251 9, .373 26. .203 -2. ,902 1. ,00 13. .76 A C
ATOM 1862 C GLY A 251 8, .026 26, .058 -2. ,248 1. ,00 14. .49 A C
ATOM 1863 O GLY A 251 6. .984 26, .057 -2. ,933 1. ,00 13. ,60 A O
ATOM 1864 N GLY A 252 8. .056 25, ,926 -0. ,920 1. 00 12. ,84 A N
ATOM 1865 CA GLY A 252 6, .879 25, .694 -0. ,101 1. ,00 13. ,22 A C
ATOM 1866 C GLY A 252 7, .242 24, .765 1. 058 1. 00 12. ,42 A C
ATOM 1867 O GLY A 252 8. .354 24, .185 1. 073 1. 00 11. 46 A O
ATOM 1868 N THR A 253 6. .328 24, .598 2. 008 1. 00 12. 37 A N ATOM 1869 CA THR A 253 6.518 23.583 3,, 043 1.00 12.51 A C
ATOM 1870 CB THR A 253 5 .543 23 .697 4 , .256 1 .00 13 .05 A C
ATOM 1871 OGl THR A 253 4 .138 23 .788 3, .858 1 .00 11 .78 A O
ATOM 1872 CG2 THR A 253 5 .837 24 .964 5, .042 1, .00 13 .23 A C
ATOM 1873 C THR A 253 6 .463 22 .211 2. .396 1 .00 12 .60 A C
ATOM 1874 O THR A 253 6 .945 21 .239 2 , .966 1 .00 12 .54 A O
ATOM 1875 N SER A 254 5 .902 22 .158 1, , 187 1 .00 12 .69 A N
ATOM 1876 CA SER A 254 5 .905 20 .957 0, .357 1 .00 12 .54 A C
ATOM 1877 CB SER A 254 5, .228 21 .233 -0. .994 1, .00 12 .41 A C
ATOM 1878 OG SER A 254 3 .822 21 .002 -0, .960 1, .00 11 .90 A O
ATOM 1879 C SER A 254 7, .298 20 .445 0. ,050 1, .00 12 .52 A c
ATOM 1880 O SER A 254 7, .459 19 .253 -0. .150 1, .00 12 .45 A 0
ATOM 1881 N MET A 255 8, .255 21 .361 -0. .054 1, .00 12 .52 A N
ATOM 1882 CA MET A 255 9, .640 21 .062 -0, .385 1, .00 13 .23 A C
ATOM 1883 CB MET A 255 10 .260 22 .231 -1 .164 1 .00 13 .16 A C
ATOM 1884 CG MET A 255 9 .955 22 .255 -2, .667 1 .00 13 .61 A C
ATOM 1885 SD MET A 255 8 .220 22 .693 -3, .027 1 .00 16 .25 A S
ATOM 1886 CE MET A 255 7 .683 21 .071 -3, .591 1 .00 13 .35 A C
ATOM 1887 C MET A 255 10 .478 20 .759 0, .873 1 .00 13 .32 A C
ATOM 1888 O MET A 255 11 .396 19 .934 0, .847 1 .00 13 .21 A O
ATOM 1889 N ALA A 256 10 .162 21 .415 1, .981 1 .00 12 .98 A N
ATOM 1890 CA ALA A 256 10 .904 21 .161 3, .213 1 .00 12 .47 A C
ATOM 1891 CB ALA A 256 10 .516 22 .175 4, .265 1 .00 11 .99 A C
ATOM 1892 C ALA A 256 10 .645 19 .737 3, .717 1 .00 11 .89 A C
ATOM 1893 O ALA A 256 11 .553 19 .018 4, .179 1 .00 11 .48 A O
ATOM 1894 N THR A 257 9 .390 19 .341 3, .629 1 .00 11 .55 A N
ATOM 1895 CA THR A 257 8 .944 18 .065 4, .146 1 .00 11 .50 A C
ATOM 1896 CB THR A 257 7 .423 17 .938 3. .908 1 .00 12 .07 A C
ATOM 1897 OGl THR A 257 6 .754 19 .013 4. .569 1 .00 13 .08 A O
ATOM 1898 CG2 THR A 257 6 .838 16 .661 4, .540 1 .00 12 .46 A C
ATOM 1899 C THR A 257 9 .705 16 .849 3. .587 1 .00 11 .20 A C
ATOM 1900 O THR A 257 10 .172 16 .018 4, .382 1 .00 11 .03 A O
ATOM 1901 N PRO A 258 9 .781 16 .686 2 , ,259 1, .00 11 .22 A N
ATOM 1902 CA PRO A 258 10 .466 15 .521 1, .687 1, .00 10 .92 A C
ATOM 1903 CB PRO A 258 10 .200 15 .644 0, .182 1, .00 10 .45 A C
ATOM 1904 CG PRO A 258 9 .884 17 .057 -0, ,029 1, .00 11 .62 A C
ATOM 1905 CD PRO A 258 9 .164 17 .504 1, .207 1, .00 10 .92 A C
ATOM 1906 C PRO A 258 11 .969 15 .503 1. .976 1, .00 10 .83 A C
ATOM 1907 O PRO A 258 12, .524 14 .417 2, ,020 1, .00 9 .95 A O
ATOM 1908 N ILE A 259 12, .605 16 .665 2. .160 1, .00 11 .19 A N
ATOM 1909 CA ILE A 259 14, .004 16 .711 2. .597 1, .00 11 .51 A C
ATOM 1910 CB ILE A 259 14, .439 18 .183 2. .712 1, .00 11 .81 A C
ATOM 1911 CGI ILE A 259 14, .492 18 .843 1. .314 1, ,00 14 .15 A C
ATOM 1912 CDl ILE A 259 15. .690 18 .403 0. .504 1, .00 17 .31 A C
ATOM 1913 CG2 ILE A 259 15. .790 18 .313 3. .375 1. .00 11 .02 A C
ATOM 1914 C ILE A 259 14, .147 15 .975 3. , 950 1, .00 11 .56 A C
ATOM 1915 O ILE A 259 15, .038 15 .133 4. .124 1, ,00 11 .81 A O
ATOM 1916 N VAL A 260 13, .259 16 .295 4. ,886 1, .00 11 .02 A N
ATOM 1917 CA VAL A 260 13, .244 15 .668 6. ,199 1. .00 12 .26 A C
ATOM 1918 CB VAL A 260 12 , ,301 16 .412 7. ,150 1, .00 12 .30 A C
ATOM 1919 CGI VAL A 260 12, .286 15, .743 8. ,557 1. ,00 12 ,78 A C
ATOM 1920 CG2 VAL A 260 12. .721 17, .855 7. ,268 1. .00 13, .51 A c
ATOM 1921 C VAL A 260 12. .847 14 , .185 6. ,106 1. .00 12, .24 A c
ATOM 1922 O VAL A 260 13. .412 13, .339 6. ,786 1. .00 12 , .79 A 0
ATOM 1923 N ALA A 261 11. ,922 13, .864 5. ,217 1. ,00 12, .41 A N
ATOM 1924 CA ALA A 261 11. .530 12, .480 4. ,997 1. ,00 11, ,93 A C
ATOM 1925 CB ALA A 261 10. .426 12 , .376 3. ,920 1. ,00 12 , ,16 A C
ATOM 1926 C ALA A 261 12. .750 11, ,661 4. ,585 1. ,00 11, .91 A C
ATOM 1927 O ALA A 261 12. ,943 10, .560 5. ,055 1. .00 11, .34 A O
ATOM 1928 N GLY A 262 13. .550 12, .186 3. ,665 1. .00 12, .22 A N
ATOM 1929 CA GLY A 262 14. .794 11, ,533 3. ,291 1. ,00 12, .29 A C
ATOM 1930 C GLY A 262 15. .786 11 .431 4. .447 1, .00 12 .34 A C
ATOM 1931 O GLY A 262 16, .414 10 .386 4. .660 1, .00 11 .90 A O ATOM 1932 N ASN A 263 15.901 12.490 5.243 1.00 12.20 A N
ATOM 1933 CA ASN A 263 16 .744 12 .433 6 .435 1 .00 11 .84 A C
ATOM 1934 CB ASN A 263 16 .772 13 .773 7 .170 1 .00 12 .31 A C
ATOM 1935 CG ASN A 263 17 .389 14 .887 6 .351 1 .00 13 .12 A C
ATOM 1936 ODl ASN A 263 18 .326 14 .681 5 .525 1 .00 15 .95 A O
ATOM 1937 ND2 ASN A 263 16 .924 16 .073 6 .600 1, .00 9 .15 A N
ATOM 1938 C ASN A 263 16 .289 11 .348 7 .396 1, .00 11 .83 A C
ATOM 1939 O ASN A 263 17 .112 10 .672 8 .020 1, .00 11 .88 A O
ATOM 1940 N VAL A 264 14, .983 11 .181 7 .517 1, .00 11 .77 A N
ATOM 1941 CA VAL A 264 14, .425 10 .138 8 .367 1, .00 12 .33 A C
ATOM 1942 CB VAL A 264 12, .893 10 .268 8, .506 1, .00 12 .44 A C
ATOM 1943 CGI VAL A 264 12, .280 9 .045 9, .178 1, .00 12 .44 A C
ATOM 1944 CG2 VAL A 264 12. .543 11 .471 9, .323 1, .00 13 .22 A C
ATOM 1945 C VAL A 264 14. .817 8 .754 7, .843 1, .00 11 .95 A C
ATOM 1946 O VAL A 264 15 .164 7 .896 8 .625 1 .00 12 .24 A O
ATOM 1947 N ALA A 265 14 .813 8 .553 6 .527 1 .00 12 .03 A N
ATOM 1948 CA ALA A 265 15 .279 7 .292 5 .966 1 .00 11 .47 A C
ATOM 1949 CB ALA A 265 15 .018 7 .237 4 .460 1 .00 11 .96 A C
ATOM 1950 C ALA A 265 16 .746 7 .046 6 .293 1, .00 11 .77 A C
ATOM 1951 O ALA A 265 17. .139 5 .932 6, .592 1, .00 11 .77 A O
ATOM 1952 N GLN A 266 17, .571 8 .091 6, .262 1. .00 12 .48 A N
ATOM 1953 CA GLN A 266 18, .999 7 .940 6, .586 1, .00 11 .99 A C
ATOM 1954 CB GLN A 266 19, .782 9 .230 6, .311 1, .00 10 .90 A C
ATOM 1955 CG GLN A 266 19, .786 9 .691 4, .865 1, .00 12 .48 A C
ATOM 1956 CD GLN A 266 20, .548 11 .011 4, .671 1, .00 12 .24 A C
ATOM 1957 OEl GLN A 266 21, .762 11 .028 4, ,352 1, .00 16 .02 A O
ATOM 1958 NE2 GLN A 266 19, .857 12 .088 4, ,853 1, .00 8 .53 A N
ATOM 1959 C GLN A 266 19, .159 7 .571 8, .046 1, .00 12 .14 A C
ATOM 1960 O GLN A 266 19, .927 6 .688 8, .398 1, .00 12 .13 A O
ATOM 1961 N LEU A 267 18, .463 8 .305 8, .898 1, .00 12 .44 A N
ATOM 1962 CA LEU A 267 18. .473 8 .049 10, .317 1, .00 12 .06 A C
ATOM 1963 CB LEU A 267 17, .624 9 .107 11, .014 1, .00 12 .45 A C
ATOM 1964 CG LEU A 267 17, .550 9 .097 12, .540 1, .00 12 .15 A C
ATOM 1965 CDl LEU A 267 18, .918 9 .293 13, .116 1, .00 12 .99 A C
ATOM 1966 CD2 LEU A 267 16, .616 10 .187 13, .009 1, .00 12 .84 A C
ATOM 1967 C LEU A 267 17, .984 6 .649 10, .654 1, .00 12 .72 A C
ATOM 1968 O LEU A 267 18, .581 5 .972 11, .497 1, .00 12 .91 A O
ATOM 1969 N ARG A 268 16, .872 6 .219 10, .044 1. .00 12 .54 A N
ATOM 1970 CA ARG A 268 16, .295 4 .886 10, .321 1 , ,00 12 .31 A C
ATOM 1971 CB ARG A 268 14, .961 4 .722 9, ,577 1 , .00 12 .21 A C
ATOM 1972 CG ARG A 268 14, .016 3 .635 10, .155 1. .00 12 .56 A C
ATOM 1973 CD ARG A 268 12. ,652 3 .605 9. .510 1. .00 14 .20 A C
ATOM 1974 NE ARG A 268 11. .781 2 .591 10. .105 1. .00 14 .70 A N
ATOM 1975 CZ ARG A 268 11. .837 1 .306 9. .829 1. .00 14, .81 A C
ATOM 1976 NHl ARG A 268 12. .697 0 .829 8. .942 1. .00 14, .11 A N
ATOM 1977 NH2 ARG A 268 10, .993 0 .483 10, .432 1, .00 15 .99 A N
ATOM 1978 C ARG A 268 17, .284 3 .763 9, .929 1. .00 12 .37 A C
ATOM 1979 O ARG A 268 17, .533 2 .837 10, .689 1, ,00 11 .86 A O
ATOM 1980 N GLU A 269 17, .846 3 .870 8, ,729 1, .00 11 .97 A N
ATOM 1981 CA GLU A 269 18, .965 3 .026 8, .306 1. .00 12 .12 A C
ATOM 1982 CB GLU A 269 19, ,561 3 .537 6, .993 1. .00 11 .36 A C
ATOM 1983 CG GLU A 269 20. .764 2 .715 6, .542 1. .00 12 .78 A C
ATOM 1984 CD GLU A 269 21. .477 3 .260 5. .335 1. .00 15 .24 A C
ATOM 1985 OEl GLU A 269 21. .277 4 .447 5. .007 1. .00 16, .05 A O
ATOM 1986 OE2 GLU A 269 22. .246 2 .479 4. .711 1. ,00 16, .25 A O
ATOM 1987 C GLU A 269 20. .082 2 .954 9. ,354 1. ,00 12 , .56 A C
ATOM 1988 O GLU A 269 20. .596 1, .875 9. .645 1. ,00 12 , .26 A O
ATOM 1989 N HIS A 270 20. ,482 4 , .104 9. .894 1. ,00 12, .79 A N
ATOM 1990 CA HIS A 270 21. ,556 4, .119 10. .859 1. ,00 12, .79 A C
ATOM 1991 CB HIS A 270 21. ,918 5, .531 11. ,289 1. ,00 12, .76 A C
ATOM 1992 CG ] BHIS A 270 23. ,160 5, .583 12. ,120 0. ,50 10, .01 A C
ATOM 1993 CG . AHIS A 270 23, .195 5 .601 12, ,063 0. .50 15 .58 A C
ATOM 1994 ND1BHIS A 270 23, .186 6 .137 13, .385 0, .50 7 .23 A N ATOM 1995 ND1AHIS A 270 23..243 5,.459 13..432 0,.50 20,.08 A N
ATOM 1996 CE1BHIS A 270 24, .404 6 .019 13. .885 0, .50 6, .26 A C
ATOM 1997 CE1AHIS A 270 24, .498 5, .548 13. .839 0. .50 20, ,34 A C
ATOM 1998 NE2BHIS A 270 25, .163 5 .387 13. .000 0, .50 8. .61 A N
ATOM 1999 NE2AHIS A 270 25. .265 5 .744 12. .783 0, .50 20, .61 A N
ATOM 2000 CD2BHIS A 270 24, .405 5 .102 11. .888 0, .50 6, .18 A C
ATOM 2001 CD2AHIS A 270 24, .475 5 .782 11. .659 0, .50 18 , ,53 A C
ATOM 2002 C HIS A 270 21, ,210 3 .294 12. ,099 1. .00 12, .75 A C
ATOM 2003 O HIS A 270 22, .031 2 .541 12. ,562 1, .00 12, .88 A O
ATOM 2004 N PHE A 271 20, .009 3 .468 12. .666 1, .00 12 , .25 A N
ATOM 2005 CA PHE A 271 19, .642 2 , .680 13. .834 1, .00 12, .36 A C
ATOM 2006 CB PHE A 271 18, .274 3 .114 14. .370 1, .00 12, .01 A C
ATOM 2007 CG PHE A 271 18. .328 4 , .292 15. ,293 1, .00 11, .54 A C
ATOM 2008 CDl PHE A 271 18, ,557 4, .127 16. ,643 1, .00 12, .44 A C
ATOM 2009 CEl PHE A 271 18 .601 5 .229 17, ,500 1 .00 13 .55 A C
ATOM 2010 CZ PHE A 271 18 .400 6 .479 17, .016 1 .00 11 .92 A C
ATOM 2011 CE2 PHE A 271 18 .145 6 .655 15, .663 1 .00 15 .14 A C
ATOM 2012 CD2 PHE A 271 18 .096 5 .567 14, .820 1 .00 13 .90 A C
ATOM 2013 C PHE A 271 19 .620 1 .178 13, ,492 1 .00 13 .05 A C
ATOM 2014 O PHE A 271 20 .147 0 .341 14, .240 1 .00 15 .06 A 0
ATOM 2015 N VAL A 272 19 .007 0 .850 12, .371 1 .00 12 .88 A N
ATOM 2016 CA VAL A 272 18 .765 -0 .526 11, .961 1 .00 13 .79 A C
ATOM 2017 CB VAL A 272 17 .856 -0 .539 10, .706 1 .00 13 .45 A C
ATOM 2018 CGI VAL A 272 17 .977 -1 .840 9, .953 1 .00 15 .25 A C
ATOM 2019 CG2 VAL A 272 16 .429 -0 .264 11, ,112 1 .00 13 .55 A C
ATOM 2020 C VAL A 272 20 .068 -1 .276 11. .689 1 .00 14 .17 A C
ATOM 2021 0 VAL A 272 20 .242 -2 .415 12. .162 1 .00 14 .67 A O
ATOM 2022 N LYS A 273 20 .992 -0 .619 10. .990 1 .00 14 .25 A N
ATOM 2023 CA LYS A 273 22 .255 -1 .217 10. .606 1 .00 14 .60 A C
ATOM 2024 CB LYS A 273 22 .759 -0 .664 9. .267 1 .00 14 .93 A C
ATOM 2025 CG LYS A 273 21 .893 -1 .085 8, ,052 1 .00 15 .97 A C
ATOM 2026 CD LYS A 273 22 .432 -0 .488 6. .729 1 .00 14, .97 A C
ATOM 2027 CE LYS A 273 21 .735 -1 .010 5, .482 1 .00 16 .43 A C
ATOM 2028 NZ LYS A 273 22 .131 -0 .162 4. .300 1 .00 13 .02 A N
ATOM 2029 C LYS A 273 23 .366 -1 .133 11, ,645 1 .00 14 .97 A C
ATOM 2030 O LYS A 273 24 .172 -2 .075 11, .740 1 .00 12 .32 A O
ATOM 2031 N ASN A 274 23 .402 -0 .033 12, .403 1 .00 14 .72 A N
ATOM 2032 CA ASN A 274 24 .556 0 .298 13, ,225 1 .00 15 .62 A C
ATOM 2033 CB ASN A 274 25 .197 1 .649 12. .786 1 .00 16 .07 A C
ATOM 2034 CG ASN A 274 25 .555 1 .662 11, .290 1 .00 17 .59 A C
ATOM 2035 ODl ASN A 274 25 .285 2 .647 10, ,543 1 .00 18 .70 A O
ATOM 2036 ND2 ASN A 274 26 .124 0 .561 10, .839 1 .00 13 .05 A N
ATOM 2037 C ASN A 274 24 .253 0 .365 14, .694 1 .00 15 .60 A C
ATOM 2038 0 ASN A 274 25 .165 0 .449 15, .465 1 .00 15 .70 A O
ATOM 2039 N ARG A 275 22 .979 0 .348 15, .092 1 .00 14 .80 A N
ATOM 2040 CA ARG A 275 22 .670 0 .517 16, .505 1 .00 15 .19 A C
ATOM 2041 CB ARG A 275 22 .046 1 .883 16, .723 1 .00 14 .89 A C
ATOM 2042 CG ARG A 275 22 .925 3 .001 16, ,141 1 .00 17 .88 A C
ATOM 2043 CD ARG A 275 22 .682 4 .354 16, .748 1 .00 17 .97 A C
ATOM 2044 NE ARG A 275 23 .098 4 .391 18, .146 1 .00 15 .44 A N
ATOM 2045 CZ ARG A 275 22 .783 5 .383 18, .977 1 .00 18 .17 A C
ATOM 2046 NHl ARG A 275 22 .080 6 .422 18. .540 1 .00 17 .27 A N
ATOM 2047 NH2 ARG A 275 23, .191 5 .356 20, ,239 1 .00 17, .72 A N
ATOM 2048 C ARG A 275 21 .796 -0 .573 17, .088 1 .00 14 .64 A C
ATOM 2049 O ARG A 275 21 .382 -0 .456 18. .212 1 .00 15, .75 A O
ATOM 2050 N GLY A 276 21 .459 -1 .577 16. .283 1, .00 14, .66 A N
ATOM 2051 CA GLY A 276 20 .880 -2 .825 16, .771 1, .00 14, .63 A C
ATOM 2052 C GLY A 276 19 .403 -2 .811 17, .060 1 .00 14, .40 A C
ATOM 2053 O GLY A 276 18 .863 -3 .751 17. .664 1 .00 13, .52 A O
ATOM 2054 N VAL A 277 18 .729 -1 .745 16. .638 1 .00 14, .10 A N
ATOM 2055 CA VAL A 277 17 .318 -1 .618 16. .894 1 .00 14, .10 A C
ATOM 2056 CB VAL A 277 17 .021 -0 .657 18 .097 1 .00 14 .71 A C
ATOM 2057 CGI VAL A 277 17 .768 -1 .058 19 .354 1 .00 14 .49 A C ATOM 2058 CG2 VAL A 277 17.268 0.771 17.733 1.00 15.67 A C
ATOM 2059 C VAL A 277 16 .547 -1 .097 15 .689 1 .00 13 .79 A C
ATOM 2060 O VAL A 277 17 .082 -0 .372 14 .853 1 .00 14 .29 A O
ATOM 2061 N THR A 278 15 .273 -1 .472 15 .607 1 .00 14 .21 A N
ATOM 2062 CA THR A 278 14 .325 -0 .778 14 .749 1 .00 14 .50 A C
ATOM 2063 CB THR A 278 13 .187 -1 .700 14 .301 1 .00 15 .23 A C
ATOM 2064 OGl THR A 278 13 .744 -2 .825 13 .607 1 .00 19 .10 A O
ATOM 2065 CG2 THR A 278 12 .304 -0 .986 13 .245 1 .00 17 .53 A C
ATOM 2066 C THR A 278 13 .760 0 .394 15 .526 1 .00 13 .49 A C
ATOM 2067 O THR A 278 13 .028 0 .210 16 .485 1 .00 13 .51 A O
ATOM 2068 N PRO A 279 14 .104 1 .612 15 .134 1 .00 12 .55 A N
ATOM 2069 CA PRO A 279 13 .679 2 .803 15 .896 1 .00 11 .32 A C
ATOM 2070 CB PRO A 279 14 .520 3 .920 15 .277 1 .00 11 .71 A C
ATOM 2071 CG PRO A 279 14 .682 3 .493 13 .842 1 .00 11 .40 A C
ATOM 2072 CD PRO A 279 14 .817 1 .975 13 .895 1 .00 12 .28 A C
ATOM 2073 C PRO A 279 12 .211 3 .055 15 .672 1 .00 11 .90 A C
ATOM 2074 O PRO A 279 11 .786 3 .053 14 .516 1 .00 12 .19 A O
ATOM 2075 N LYS A 280 11 .438 3 .212 16 .743 1 .00 11 .50 A N
ATOM 2076 CA LYS A 280 10 .020 3 .518 16 .639 1 .00 12 .77 A C
ATOM 2077 CB LYS A 280 9 .354 3 .389 18 .024 1 .00 13 .46 A C
ATOM 2078 CG LYS A 280 9 .324 1 .993 18 .573 1 .00 15 .21 A C
ATOM 2079 CD LYS A 280 8 .273 1 .192 17, .861 1 .00 20 .42 A C
ATOM 2080 CE LYS A 280 8 .012 -0 .146 18, .555 1 .00 23 .45 A C
ATOM 2081 NZ LYS A 280 6 .935 -0 .858 17, ,808 1 .00 26 .12 A N
ATOM 2082 C LYS A 280 9 .811 4 .951 16 .120 1 .00 11 .96 A C
ATOM 2083 O LYS A 280 10 .710 5 .782 16, .200 1 .00 13 .23 A O
ATOM 2084 N PRO A 281 8 .666 5 .233 15, .512 1 .00 12 .23 A N
ATOM 2085 CA PRO A 281 8 .370 6 .608 15, .073 1 .00 11 .69 A C
ATOM 2086 CB PRO A 281 6 .897 6 .540 14, .763 1, .00 12 .25 A C
ATOM 2087 CG PRO A 281 6, .755 5 .162 14, .210 1, .00 12, .66 A C
ATOM 2088 CD PRO A 281 7, .592 4 .300 15. ,126 1, ,00 11, .87 A C
ATOM 2089 C PRO A 281 8, .682 7, .678 16. ,105 1. .00 11, ,94 A C
ATOM 2090 O PRO A 281 9, ,287 8, .708 15, ,734 1. ,00 11. .60 A O
ATOM 2091 N SER A 282 8, .303 7 .447 17. .374 1, .00 11, .91 A N
ATOM 2092 CA SER A 282 8, .579 8 .404 18, .442 1, .00 11, ,78 A C
ATOM 2093 CB SER A 282 8, .017 7 .930 19. ,789 1 , .00 11, .85 A C
ATOM 2094 OG SER A 282 8, ,503 6, .639 20. ,117 1. .00 12. .30 A O
ATOM 2095 C SER A 282 10, .049 8, .704 18. ,654 1. ,00 11. ,36 A C
ATOM 2096 O SER A 282 10, .402 9, .835 19. ,014 1. ,00 11. ,10 A O
ATOM 2097 N LEU A 283 10. .896 7 , .696 18. ,498 1. ,00 11. ,81 A N
ATOM 2098 CA LEU A 283 12. .332 7 , .889 18. ,642 1. ,00 11. ,66 A C
ATOM 2099 CB LEU A 283 13. .042 6, .532 18. 856 1. .00 11. ,73 A C
ATOM 2100 CG LEU A 283 14. .575 6 .628 18. ,893 1. .00 11. .60 A C
ATOM 2101 CDl LEU A 283 15 .029 7 .501 20. .001 1, .00 10. ,08 A C
ATOM 2102 CD2 LEU A 283 15. .180 5 .233 19. .066 1. .00 15. ,86 A C
ATOM 2103 C LEU A 283 12, .953 8 .650 17. .465 1. .00 11. .15 A C
ATOM 2104 O LEU A 283 13, .812 9 .515 17. .644 1. .00 11. 72 A O
ATOM 2105 N LEU A 284 12. .575 8, .305 16. ,244 1. .00 11. 65 A N
ATOM 2106 CA LEU A 284 13. .056 9, .058 15. ,088 1. .00 10. 89 A C
ATOM 2107 CB LEU A 284 12 , ,493 8, .470 13. 802 1. ,00 10. 71 A C
ATOM 2108 CG LEU A 284 13. ,010 7 , .059 13. 442 1. ,00 10. 82 A C
ATOM 2109 CDl LEU A 284 12. .102 6 , ,419 12. 399 1. 00 10. 74 A C
ATOM 2110 CD2 LEU A 284 14, .425 7 .107 12. ,953 1. ,00 10. 79 A C
ATOM 2111 C LEU A 284 12, ,741 10, .568 15. ,245 1 , ,00 10. 24 A C
ATOM 2112 O LEU A 284 13. .591 11, .414 15. ,013 1. .00 9. 64 A O
ATOM 2113 N LYS A 285 11. .527 10, .868 15. 682 1. ,00 10. 77 A N
ATOM 2114 CA LYS A 285 11, ,054 12 , .217 15. 890 1. 00 10. 81 A C
ATOM 2115 CB LYS A 285 9. .544 12 , .188 16. 152 1. 00 10. 59 A C
ATOM 2116 CG LYS A 285 8. .909 13 , .531 16. 527 1. 00 10. 18 A C
ATOM 2117 CD LYS A 285 7. ,372 13. .380 16. 583 1. 00 12. 54 A C
ATOM 2118 CE LYS A 285 6. ,660 14. .630 17. 085 1. 00 11. 16 A C
ATOM 2119 NZ LYS A 285 5, .159 14 , .525 16. 941 1. ,00 9. 27 A N
ATOM 2120 C LYS A 285 11, .816 12 , .886 17. 037 1. ,00 10. 86 A C ATOM 2121 O LYS A 285 12.287 13.995 16.888 1.00 11.16 A O
ATOM 2122 N ALA A 286 11 .964 12 .194 18, .156 1. .00 10 .94 A N
ATOM 2123 CA ALA A 286 12 .744 12 .722 19 .280 1 .00 11 .16 A C
ATOM 2124 CB ALA A 286 12 .657 11 .813 20 .437 1 .00 11 .37 A C
ATOM 2125 C ALA A 286 14 .206 12 .952 18, .897 1 .00 10 .98 A C
ATOM 2126 O ALA A 286 14 .794 13 .947 19 .275 1 .00 10 .07 A O
ATOM 2127 N ALA A 287 14 .778 12 .048 18 .115 1 .00 11 .61 A N
ATOM 2128 CA ALA A 287 16 .175 12 .206 17 .679 1 .00 12 .13 A C
ATOM 2129 CB ALA A 287 16 .692 10 .922 17 .034 1. .00 11 .58 A C
ATOM 2130 C ALA A 287 16 .349 13 .411 16 .742 1 .00 12 .42 A C
ATOM 2131 O ALA A 287 17 .310 14 .165 16, .873 1 .00 11 .82 A O
ATOM 2132 N LEU A 288 15 .407 13 .623 15 .826 1 .00 12 .37 A N
ATOM 2133 CA LEU A 288 15 .473 14 .808 14 .956 1 .00 13 .09 A C
ATOM 2134 CB LEU A 288 14 .357 14 .775 13, .917 1, .00 13 .67 A C
ATOM 2135 CG LEU A 288 14 .552 13 .833 12, .736 1 .00 15 .39 A C
ATOM 2136 CDl LEU A 288 13 .379 14 .033 11 .840 1 .00 19 .44 A C
ATOM 2137 CD2 LEU A 288 15 .842 14 .113 11, .974 1, .00 15 .17 A C
ATOM 2138 C LEU A 288 15 .329 16 .105 15, .747 1 .00 12 .69 A C
ATOM 2139 O LEU A 288 16 .014 17 .107 15, .481 1 .00 12 .69 A O
ATOM 2140 N ILE A 289 14, .412 16 .096 16, .704 1, .00 12 .33 A N
ATOM 2141 CA ILE A 289 14 .195 17 .261 17, .546 1, .00 12 .69 A C
ATOM 2142 CB ILE A 289 12 .920 17 .084 18, .397 1, .00 12 .55 A C
ATOM 2143 CGI ILE A 289 11 .688 17 .178 17 .488 1 .00 11 .46 A C
ATOM 2144 CDl ILE A 289 10 .404 16 .725 18, ,104 1, .00 10 .00 A C
ATOM 2145 CG2 ILE A 289 12 .869 18 .098 19, .506 1, .00 13 .54 A C
ATOM 2146 C ILE A 289 15 .412 17 .596 18, .426 1 .00 12 .74 A C
ATOM 2147 O ILE A 289 15 .844 18 .746 18, .442 1, .00 13 .36 A O
ATOM 2148 N ALA A 290 15 .975 16 .612 19, .132 1, .00 11 .71 A N
ATOM 2149 CA ALA A 290 17 .132 16 .854 19, .976 1 .00 11 .73 A C
ATOM 2150 CB ALA A 290 17, .529 15 .593 20, .698 1, .00 12 .08 A C
ATOM 2151 C ALA A 290 18 .326 17 .370 19, .191 1, .00 11 .95 A C
ATOM 2152 O ALA A 290 19 .114 18 .198 19, .689 1 .00 10 .65 A O
ATOM 2153 N GLY A 291 18, .472 16 .866 17, .979 1, ,00 11 .84 A N
ATOM 2154 CA GLY A 291 19 .633 17 .207 17, .171 1, .00 12 .60 A C
ATOM 2155 C GLY A 291 19 .466 18 .506 16, .400 1, .00 12 .44 A C
ATOM 2156 O GLY A 291 20, .437 19, .018 15. .847 1, .00 11 .56 A O
ATOM 2157 N ALA A 292 18 .249 19 .056 16, ,378 1, .00 12 .96 A N
ATOM 2158 CA ALA A 292 17 .960 20 .238 15, .562 1, .00 13 .11 A C
ATOM 2159 CB ALA A 292 16. .454 20, .468 15, .434 1, .00 12 .99 A C
ATOM 2160 C ALA A 292 18, .655 21 .499 16, .075 1, .00 13 .45 A C
ATOM 2161 O ALA A 292 18 .954 21 .612 17, .252 1 .00 13 .95 A O
ATOM 2162 N ALA A 293 18. .848 22, .456 15. .173 1, ,00 13 .18 A N
ATOM 2163 CA ALA A 293 19. .567 23 .694 15, .450 1, .00 13 .43 A C
ATOM 2164 CB ALA A 293 20 .508 24 .012 14, .284 1 .00 13 .22 A C
ATOM 2165 C ALA A 293 18, .611 24, .866 15, .637 1, .00 13 .54 A C
ATOM 2166 O ALA A 293 17, .739 25 .107 14, .812 1, .00 13 .39 A O
ATOM 2167 N ASP A 294 18 .838 25 .626 16, .691 1 .00 13 .60 A N
ATOM 2168 CA ASP A 294 18, ,167 26, .895 16. .899 1, ,00 14 .04 A C
ATOM 2169 CB ASP A 294 18, .590 27 .428 18. .270 1, ,00 14 .33 A C
ATOM 2170 CG ASP A 294 17 .918 28 .728 18. ,634 1, .00 14 .83 A C
ATOM 2171 ODl ASP A 294 18, .142 29, .160 19. .799 1, .00 11 .77 A O
ATOM 2172 OD2 ASP A 294 17, .144 29, .360 17, .861 1, .00 10 .92 A O
ATOM 2173 C ASP A 294 18 .620 27 .814 15, .774 1 .00 13 .72 A C
ATOM 2174 O ASP A 294 19 .801 28 .075 15, .636 1 .00 14 .66 A O
ATOM 2175 N VAL A 295 17, .696 28 .304 14, .956 1, .00 14 .43 A N
ATOM 2176 CA VAL A 295 18, .057 29 .217 13, .853 1, .00 14 .05 A C
ATOM 2177 CB VAL A 295 16 .957 29 .283 12, .730 1 .00 13 .77 A C
ATOM 2178 CGI VAL A 295 16, .670 27 .907 12, .184 1, .00 13 .93 A C
ATOM 2179 CG2 VAL A 295 15, .680 29 .956 13, .255 1, .00 12 .53 A C
ATOM 2180 C VAL A 295 18 .352 30 .622 14, .346 1 .00 14 .82 A c
ATOM 2181 O VAL A 295 18, .707 31 .497 13, .558 1, .00 15 .77 A 0
ATOM 2182 N GLY A 296 18, .190 30 .857 15, .646 1 .00 14 .99 A N
ATOM 2183 CA GLY A 296 18 .450 32 .159 16, .208 1 .00 15 .10 A C ATOM 2184 C GLY A 296 17.282 32.821 16.906 1,.00 15.03 A C
ATOM 2185 O GLY A 296 17 .458 33 .886 17 .489 1, .00 15 .00 A O
ATOM 2186 N LEU A 297 16 .114 32 .180 16 .887 1, .00 16 .05 A N
ATOM 2187 CA LEU A 297 14 .903 32 .728 17 .501 1 .00 15 .21 A C
ATOM 2188 CB LEU A 297 13 .704 32 .420 16 .605 1 .00 14 .95 A C
ATOM 2189 CG LEU A 297 13 .881 32 .962 15 .179 1 .00 17 .47 A C
ATOM 2190 CDl LEU A 297 12 .718 32 .507 14 .330 1 .00 19 .73 A C
ATOM 2191 CD2 LEU A 297 13 .939 34 .500 15 .204 1 .00 18 .75 A C
ATOM 2192 C LEU A 297 14 .645 32 .187 18 .903 1 .00 15 .26 A C
ATOM 2193 O LEU A 297 13 .782 32 .682 19 .636 1 .00 14 .95 A O
ATOM 2194 N GLY A 298 15 .380 31 .152 19 .266 1 .00 15 .28 A N
ATOM 2195 CA GLY A 298 15 .251 30 .546 20 .570 1 .00 15 .27 A C
ATOM 2196 C GLY A 298 14 .024 29 .691 20 .721 1 .00 15 .55 A C
ATOM 2197 O GLY A 298 13 .173 29 .572 19 .817 1 .00 15 .07 A O
ATOM 2198 N PHE A 299 13 .941 29 .090 21 .894 1 .00 15 .47 A N
ATOM 2199 CA PHE A 299 12 .820 28 .269 22 .266 1 .00 16 .76 A C
ATOM 2200 CB PHE A 299 13 .279 26 .818 22 .539 1, .00 16 .33 A C
ATOM 2201 CG PHE A 299 14 .108 26 .237 21, .435 1, .00 16 .07 A C
ATOM 2202 CDl PHE A 299 13 .572 26 .047 20, .167 1, .00 15 .70 A C
ATOM 2203 CEl PHE A 299 14 .349 25 .544 19, .132 1, .00 15 .95 A C
ATOM 2204 CZ PHE A 299 15, .660 25 .233 19, .357 1, .00 15, .27 A C
ATOM 2205 CE2 PHE A 299 16, .217 25 .432 20, ,610 1. .00 15, .20 A C
ATOM 2206 CD2 PHE A 299 15, .436 25 .929 21, ,642 1. .00 17, .28 A C
ATOM 2207 C PHE A 299 12, .200 28 .872 23. .504 1, ,00 17, .91 A C
ATOM 2208 O PHE A 299 12, .886 29 .548 24. .280 1, .00 19, ,79 A O
ATOM 2209 N PRO A 300 10, .904 28 .683 23. .680 1 , .00 19. .34 A N
ATOM 2210 CA PRO A 300 10 .054 27 .945 22 .747 1 .00 20 .29 A C
ATOM 2211 CB PRO A 300 8 .819 27 .671 23, .583 1, .00 20 .94 A C
ATOM 2212 CG PRO A 300 8 .712 28 .898 24, .513 1. .00 19 .43 A C
ATOM 2213 CD PRO A 300 10 .120 29 .311 24, .758 1, .00 20. .25 A C
ATOM 2214 C PRO A 300 9 .672 28 .834 21, .581 1, .00 20, .84 A C
ATOM 2215 O PRO A 300 9 .921 30 .043 21, .655 1, .00 21, .44 A O
ATOM 2216 N ASN A 301 9 .076 28 .282 20, .524 1, ,00 21, .28 A N
ATOM 2217 CA ASN A 301 8, .874 29 .078 19, .319 1, .00 21, .53 A C
ATOM 2218 CB ASN A 301 10, .198 29 .173 18, .567 1, .00 21. .34 A C
ATOM 2219 CG ASN A 301 10 .320 30 .458 17, .764 1, .00 20. .14 A C
ATOM 2220 ODl ASN A 301 9, .875 30 .553 16, .607 1, ,00 17. .42 A O
ATOM 2221 ND2 ASN A 301 10. .920 31 .454 18. .371 1. .00 22. .84 A N
ATOM 2222 C ASN A 301 7, .823 28 .534 18, .355 1. .00 22. .14 A C
ATOM 2223 O ASN A 301 7, .868 27 .379 17, ,978 1, .00 23, .49 A O
ATOM 2224 N GLY A 302 6, .970 29 .416 17. .854 1 , .00 22, .24 A N
ATOM 2225 CA GLY A 302 5. .912 29 .031 16. .932 1, .00 21, ,65 A C
ATOM 2226 C GLY A 302 6, .292 29 .165 15, ,475 1. .00 20, .83 A C
ATOM 2227 O GLY A 302 5, .574 28, .669 14. .603 1. .00 21, .22 A O
ATOM 2228 N ASN A 303 7, .446 29, .787 15. .225 1. .00 19, .62 A N
ATOM 2229 CA ASN A 303 7, ,981 30, .005 13. .886 1, .00 17, .66 A C
ATOM 2230 CB ASN A 303 8. .705 31, .349 13. .816 1. .00 17, ,45 A C
ATOM 2231 CG ASN A 303 7, .872 32, .473 14. .381 1. .00 20, .84 A C
ATOM 2232 ODl ASN A 303 6, ,802 32, .779 13. ,847 1. ,00 21. .34 A O
ATOM 2233 ND2 ASN A 303 8 .305 33 .033 15, .530 1. .00 22. .58 A N
ATOM 2234 C ASN A 303 8 .926 28 .922 13, .414 1, .00 15, .61 A C
ATOM 2235 O ASN A 303 8 .831 28 .481 12, ,239 1, .00 13. .70 A O
ATOM 2236 N GLN A 304 9, .841 28 .506 14, .298 1, .00 14, .18 A N
ATOM 2237 CA GLN A 304 10, .855 27 .513 13, .946 1, .00 13, .13 A C
ATOM 2238 CB GLN A 304 12, .235 28 .005 14, ,316 1, .00 13, .28 A C
ATOM 2239 CG GLN A 304 12, .556 27 .976 15. .811 1, .00 12, ,80 A C
ATOM 2240 CD GLN A 304 14, .020 28 .087 16. .123 1 , .00 11, ,63 A C
ATOM 2241 OEl GLN A 304 14, .842 27 .582 15. .386 1. .00 13, .19 A O
ATOM 2242 NE2 GLN A 304 14, ,352 28 .794 17. .223 1 , .00 9, .76 A N
ATOM 2243 C GLN A 304 10, .601 26 .128 14. .582 1 , ,00 13, .52 A C
ATOM 2244 O GLN A 304 11. .372 25 .208 14. .381 1 , .00 13 , ,66 A O
ATOM 2245 N GLY A 305 9. .537 25 .972 15. .343 1 , .00 12, .81 A N
ATOM 2246 CA GLY A 305 9. .351 24 .745 16. .101 1 , .00 12, .54 A C ATOM 2247 C GLY A 305 10.578 24.498 16.970 1.00 12.99 A C
ATOM 2248 O GLY A 305 11 .062 25 .409 17 .633 1 .00 11 .36 A O
ATOM 2249 N TRP A 306 11 .107 23 .272 16 .926 1 .00 12 .12 A N
ATOM 2250 CA TRP A 306 12 .286 22 .898 17 .701 1 .00 12 .07 A C
ATOM 2251 CB TRP A 306 12, .130 21 .473 18, .254 1 .00 12 .05 A C
ATOM 2252 CG TRP A 306 10, .866 21 .349 19 .092 1 .00 11 .34 A C
ATOM 2253 CDl TRP A 306 9 .755 20 .611 18, .805 1 .00 12 .28 A C
ATOM 2254 NEl TRP A 306 8, .812 20 .766 19, .794 1 .00 13 .42 A N
ATOM 2255 CE2 TRP A 306 9, .316 21 .592 20, .761 1, .00 12 .23 A C
ATOM 2256 CD2 TRP A 306 10, .616 21 .963 20, .359 1, .00 13 .78 A C
ATOM 2257 CE3 TRP A 306 11, .344 22 .822 21, .183 1, .00 13 .41 A C
ATOM 2258 CZ3 TRP A 306 10, .780 23 .247 22, .365 1, .00 12 .96 A C
ATOM 2259 CH2 TRP A 306 9, .488 22 .854 22, .735 1, .00 13 .70 A C
ATOM 2260 CZ2 TRP A 306 8 .744 22 .035 21 .945 1 .00 14 .65 A C
ATOM 2261 C TRP A 306 13 .562 23 .046 16 .890 1 .00 12 .73 A C
ATOM 2262 O TRP A 306 14 .632 22 .620 17 .318 1 .00 14 .46 A O
ATOM 2263 N GLY A 307 13 .479 23 .716 15 .741 1 .00 13 .10 A N
ATOM 2264 CA GLY A 307 14 .679 24 .111 15 .015 1 .00 12 .18 A C
ATOM 2265 C GLY A 307 14 .871 23 .392 13 .685 1 .00 11 .91 A C
ATOM 2266 O GLY A 307 14 .008 22 .628 13 .238 1 .00 11 .20 A O
ATOM 2267 N ARG A 308 16 .007 23 .658 13 .046 1 .00 11 .60 A N
ATOM 2268 CA ARG A 308 16 .299 23 .167 11 .701 1 .00 12 .01 A C
ATOM 2269 CB ARG A 308 17 .153 24 .223 10 .961 1 .00 12 .63 A C
ATOM 2270 CG ARG A 308 17 .388 23 .970 9 .462 1 .00 13 .43 A C
ATOM 2271 CD ARG A 308 18 .284 25 .025 8 .807 1 .00 13 .61 A C
ATOM 2272 NE ARG A 308 19 .563 25 .031 9, .510 1 .00 16 .45 A N
ATOM 2273 CZ ARG A 308 20 .127 26 .086 10, .086 1 .00 16 .43 A C
ATOM 2274 NHl ARG A 308 21 .257 25 .902 10, .762 1 .00 14 .45 A N
ATOM 2275 NH2 ARG A 308 19, .660 27 .319 9, .902 1 .00 13 .23 A N
ATOM 2276 C ARG A 308 17 .055 21 .837 11, .770 1 .00 12 .38 A C
ATOM 2277 O ARG A 308 18, .094 21 .742 12, .434 1, .00 12 .82 A O
ATOM 2278 N VAL A 309 16, .549 20 .817 11, ,080 1, .00 11 .99 A N
ATOM 2279 CA VAL A 309 17, .170 19 .498 11, , 096 1, .00 12 .04 A C
ATOM 2280 CB VAL A 309 16, .553 18 .573 10, .051 1, .00 11 .35 A C
ATOM 2281 CGI VAL A 309 17, .354 17 .272 9, ,961 1, .00 11 .45 A C
ATOM 2282 CG2 VAL A 309 15, .047 18 .335 10, ,377 1, ,00 11 .38 A C
ATOM 2283 C VAL A 309 18, .667 19 .545 10. .868 1, .00 12 .28 A C
ATOM 2284 O VAL A 309 19. .148 20 .157 9. .930 1, .00 11. .94 A O
ATOM 2285 N THR A 310 19, .393 18 .904 11. .760 1, .00 13 , .11 A N
ATOM 2286 CA THR A 310 20, .854 18 .814 11. .675 1, .00 12 , .86 A C
ATOM 2287 CB THR A 310 21. .471 19 .719 12. .723 1, .00 13 .26 A C
ATOM 2288 OGl THR A 310 21, .044 21 .090 12. .516 1, ,00 12 .98 A O
ATOM 2289 CG2 THR A 310 22 , .962 19 .747 12. .610 1, .00 13 .68 A C
ATOM 2290 C THR A 310 21, .197 17 .347 11. .911 1, .00 13 .94 A C
ATOM 2291 O THR A 310 21, .411 16 .896 13. .064 1, .00 14 .93 A O
ATOM 2292 N LEU A 311 21, .269 16 .601 10. .818 1, .00 13, .28 A N
ATOM 2293 CA LEU A 311 21, .110 15 .149 10. .879 1, ,00 14 , .11 A C
ATOM 2294 CB LEU A 311 20. .956 14 .589 9. ,463 1. .00 13, .67 A C
ATOM 2295 CG LEU A 311 20. .879 13 .078 9. ,311 1, .00 13, .50 A C
ATOM 2296 CDl LEU A 311 19. .749 12 .522 10. ,109 1. .00 15, ,11 A C
ATOM 2297 CD2 LEU A 311 20. .749 12 .688 7. ,849 1. .00 14, .22 A C
ATOM 2298 C LEU A 311 22. .252 14, .455 11. ,605 1. .00 14, .58 A C
ATOM 2299 O LEU A 311 22. .012 13 .521 12. ,345 1. ,00 14, .74 A O
ATOM 2300 N ASP A 312 23. .483 14 .959 11. ,462 1. .00 15. .58 A N
ATOM 2301 CA ASP A 312 24. ,624 14, ,307 12. , 111 1. .00 16. .68 A C
ATOM 2302 CB ASP A 312 25. .984 14, ,797 11. ,582 1. ,00 16, ,93 A C
ATOM 2303 CG ASP A 312 26. .222 16, .245 11. ,822 1. ,00 19, .05 A C
ATOM 2304 ODl ASP A 312 27. .394 16, .614 11. ,755 1. .00 25, .94 A O
ATOM 2305 OD2 ASP A 312 25. .348 17, .090 12. ,090 1. .00 18. .31 A O
ATOM 2306 C ASP A 312 24, .560 14, .330 13. ,615 1. ,00 16. .48 A C
ATOM 2307 O ASP A 312 24, .965 13 .381 14. .241 1. ,00 17 .40 A O
ATOM 2308 N LYS A 313 23, .976 15 .364 14, .202 1, ,00 16 .32 A N
ATOM 2309 CA LYS A 313 23, .755 15 .366 15. .641 1, .00 16 .35 A C ATOM 2310 CB LYS A 313 23.363 16.773 16.130 1.00 17.66 A C
ATOM 2311 CG LYS A 313 24 .430 17 .848 15 .938 1 .00 20 .65 A C
ATOM 2312 CD LYS A 313 24 .735 18 .590 17 .250 1 .00 28 .29 A C
ATOM 2313 CE LYS A 313 25 .619 17 .746 18 .162 1 .00 31 .10 A C
ATOM 2314 NZ LYS A 313 26 .458 18 .529 19 .131 1 .00 32 .31 A N
ATOM 2315 C LYS A 313 22 .666 14 .379 16 .100 1 .00 15 .36 A C
ATOM 2316 O LYS A 313 22 .662 13 .968 17 .258 1 .00 15 .49 A O
ATOM 2317 N SER A 314 21 .755 14 .007 15 .212 1 .00 14 .12 A N
ATOM 2318 CA SER A 314 20 .700 13 .047 15 .537 1 .00 13 .37 A C
ATOM 2319 CB SER A 314 19 .497 13 .217 14 .590 1 .00 13 .56 A C
ATOM 2320 OG SER A 314 18 .889 14 .489 14 .723 1 .00 11 .16 A O
ATOM 2321 C SER A 314 21 .148 11 .581 15 .505 1 .00 14 .53 A C
ATOM 2322 O SER A 314 20 .507 10 .717 16 .112 1 .00 14 .30 A O
ATOM 2323 N LEU A 315 22 .209 11 .285 14 .771 1 .00 14 .58 A N
ATOM 2324 CA LEU A 315 22 .563 9 .896 14 .484 1 .00 15 .64 A C
ATOM 2325 CB LEU A 315 23 .750 9 .824 13 .505 1 .00 15 .15 A C
ATOM 2326 CG LEU A 315 23 .506 10 .436 12, .143 1 .00 14 .88 A C
ATOM 2327 CDl LEU A 315 24 .834 10 .445 11, .360 1 .00 16 .67 A C
ATOM 2328 CD2 LEU A 315 22. .401 9 .683 11, .411 1 .00 16 .28 A C
ATOM 2329 C LEU A 315 22, .905 9 .095 15. .733 1, .00 16, .16 A C
ATOM 2330 O LEU A 315 22, .442 7. .956 15. .894 1, .00 16, .79 A O
ATOM 2331 N ASN A 316 23, .692 9, .686 16, ,631 1, .00 17, .60 A N
ATOM 2332 CA ASN A 316 24, .235 8. .915 17. .749 1, .00 17, .99 A C
ATOM 2333 CB ASN A 316 25, .757 8, .837 17. .694 1, .00 19, .59 A C
ATOM 2334 CG ASN A 316 26, .264 7 , ,879 16. .601 1, .00 22. .56 A C
ATOM 2335 ODl ASN A 316 25, .736 6, .732 16. .397 1, .00 21. .64 A O
ATOM 2336 ND2 ASN A 316 27 .321 8 .320 15. .910 1 .00 23 .13 A N
ATOM 2337 C ASN A 316 23 .732 9 .396 19. .116 1 .00 17 .61 A C
ATOM 2338 O ASN A 316 24, .390 9 .212 20, , 152 1, .00 15. .93 A O
ATOM 2339 N VAL A 317 22, .505 9, .912 19, .123 1, .00 15, .75 A N
ATOM 2340 CA VAL A 317 21, .820 10, .220 20, .389 1. .00 15, .40 A C
ATOM 2341 CB VAL A 317 20, .360 10, .675 20. .150 1, .00 15, .21 A C
ATOM 2342 CGI VAL A 317 20, ,335 11, .940 19. .350 1, .00 16, ,28 A C
ATOM 2343 CG2 VAL A 317 19, .547 9, .600 19. .458 1, .00 16, .07 A C
ATOM 2344 C VAL A 317 21, .803 8, .995 21. .323 1, .00 14 , .67 A C
ATOM 2345 O VAL A 317 21, .675 7, .864 20. .868 1, .00 14, ,77 A O
ATOM 2346 N ALA A 318 21, ,932 9, .225 22. .627 1, .00 13 , .95 A N
ATOM 2347 CA ALA A 318 21. .623 8 , .186 23. .603 1, .00 13. .64 A C
ATOM 2348 CB ALA A 318 22. .196 8. .523 24. ,940 1, .00 13. .50 A C
ATOM 2349 C ALA A 318 20. .087 8, .130 23. .652 1, .00 13. ,19 A C
ATOM 2350 O ALA A 318 19. .416 9, ,168 23. .498 1, .00 13. .46 A O
ATOM 2351 N PHE A 319 19, .512 6, .952 23. ,853 1, .00 12. .88 A N
ATOM 2352 CA PHE A 319 18, .076 6, .824 23. .633 1. .00 12. .66 A C
ATOM 2353 CB PHE A 319 17. .815 6, ,531 22. .137 1. .00 12. .79 A C
ATOM 2354 CG PHE A 319 18. .267 5, .158 21. ,700 1, .00 14. .03 A C
ATOM 2355 CDl PHE A 319 19, .504 4 , .980 21. ,107 1. ,00 12. .44 A C
ATOM 2356 CEl PHE A 319 19. .930 3 , .725 20. ,718 1. .00 15. .58 A C
ATOM 2357 CZ PHE A 319 19. .115 2 , .619 20. ,921 1. .00 15. .26 A C
ATOM 2358 CE2 PHE A 319 17. .887 2 , .777 21. ,524 1. ,00 14. ,77 A C
ATOM 2359 CD2 PHE A 319 17, .461 4 .047 21, .905 1. .00 15, .05 A C
ATOM 2360 C PHE A 319 17, .349 5 .799 24, .461 1. .00 12, .38 A C
ATOM 2361 O PHE A 319 17, .947 4 .918 25, .055 1, .00 12 , .11 A O
ATOM 2362 N VAL A 320 16 .032 5 .947 24, .488 1. .00 12 , ,04 A N
ATOM 2363 CA VAL A 320 15, .125 4 .909 24, .949 1. .00 12, .42 A C
ATOM 2364 CB VAL A 320 14, .391 5, .323 26, .252 1, .00 13, .15 A C
ATOM 2365 CGI VAL A 320 13, .237 4 .331 26. .589 1, .00 13, ,46 A c
ATOM 2366 CG2 VAL A 320 15, ,401 5, .395 27, .416 1, .00 13, .91 A c
ATOM 2367 C VAL A 320 14, .137 4 .758 23. .802 1. .00 11, .88 A c
ATOM 2368 O VAL A 320 13, .668 5, .749 23, .271 1, .00 10. .13 A 0
ATOM 2369 N ASN A 321 13, .824 3, .520 23. .441 1, ,00 11. .82 A N
ATOM 2370 CA ASN A 321 13, ,018 3, .205 22. .261 1. .00 11, .75 A C
ATOM 2371 CB ASN A 321 13, .858 2 .323 21. .313 1, .00 11, .75 A C
ATOM 2372 CG ASN A 321 13. .214 2 , .117 19. .944 1. .00 12. .10 A C ATOM 2373 ODl ASN A 321 12.506 3.005 19.437 1.00 12.86 A O
ATOM 2374 ND2 ASN A 321 13 .451 0 .919 19 .328 1 .00 10 .43 A N
ATOM 2375 C ASN A 321 11 .711 2 .463 22 .637 1 .00 12 .63 A C
ATOM 2376 O ASN A 321 11 .509 1 .311 22 .260 1 .00 12 .41 A O
ATOM 2377 N GLU A 322 10 .835 3 .130 23 .380 1 .00 12 .99 A N
ATOM 2378 CA GLU A 322 9 .542 2 .552 23 .760 1 .00 13 .23 A C
ATOM 2379 CB GLU A 322 8 .601 2 .415 22 .528 1 .00 13 .10 A C
ATOM 2380 CG GLU A 322 8 .146 3 .794 22 .013 1 .00 12 .18 A C
ATOM 2381 CD GLU A 322 7 .072 3 .781 20 .933 1 .00 14 .48 A C
ATOM 2382 OEl GLU A 322 6 .747 4 .884 20 .429 1 .00 15 .20 A O
ATOM 2383 OE2 GLU A 322 6 .564 2 .690 20 .588 1 .00 14 .00 A O
ATOM 2384 C GLU A 322 9 .654 1 .239 24 .556 1 .00 14 .04 A C
ATOM 2385 O GLU A 322 8 .778 0 .385 24 .468 1 .00 12 .70 A O
ATOM 2386 N THR A 323 10 .688 1 .122 25 .387 1 .00 13 .33 A N
ATOM 2387 CA THR A 323 10 .907 -0 .101 26 .145 1 .00 14 .72 A C
ATOM 2388 CB THR A 323 12 .375 -0 .466 26 .173 1 .00 14 .45 A C
ATOM 2389 OGl THR A 323 13 .168 0 .721 26 .401 1 .00 16 .74 A O
ATOM 2390 CG2 THR A 323 12 .813 -0 .980 24 .810 1 .00 15 .39 A C
ATOM 2391 C THR A 323 10 .397 -0 .045 27 .589 1 .00 15 .63 A C
ATOM 2392 O THR A 323 10 .572 -1 .011 28 .319 1 .00 14 .71 A 0
ATOM 2393 N SER A 324 9 .796 1 .073 28 .002 1 .00 15 .41 A N
ATOM 2394 CA SER A 324 9, .176 1 .159 29 .341 1 .00 15 .78 A C
ATOM 2395 CB SER A 324 10, .008 2 .045 30, .272 1 .00 15 .94 A C
ATOM 2396 OG SER A 324 11, .281 1 .460 30, .594 1 .00 17 .86 A O
ATOM 2397 C SER A 324 7, .739 1 .723 29, .257 1 .00 15 .74 A C
ATOM 2398 O SER A 324 7, ,558 2 .937 29, .315 1 .00 15 .93 A 0
ATOM 2399 N PRO A 325 6 .729 0 .864 29 .118 1 .00 16 .08 A N
ATOM 2400 CA PRO A 325 5 .326 1 .306 29 .170 1 .00 17 .17 A C
ATOM 2401 CB PRO A 325 4 .525 0 .035 28 .823 1 .00 17 .76 A C
ATOM 2402 CG PRO A 325 5, .454 -1 .106 29 .084 1 .00 17 .45 A C
ATOM 2403 CD PRO A 325 6 .845 -0 .585 28 .885 1 .00 16 .38 A C
ATOM 2404 C PRO A 325 4, .926 1 .815 30 .548 1 .00 17 .68 A C
ATOM 2405 O PRO A 325 5, .277 1 .211 31, .553 1 .00 18 .68 A O
ATOM 2406 N LEU A 326 4, .204 2 .916 30, ,596 1. .00 18 .30 A N
ATOM 2407 CA LEU A 326 3, .796 3 .491 31. .871 1, .00 18 .53 A C
ATOM 2408 CB LEU A 326 4, .410 4 .890 32, .059 1, .00 18 .37 A C
ATOM 2409 CG LEU A 326 5, .938 5 .027 32, ,161 1, .00 19 .30 A C
ATOM 2410 CDl LEU A 326 6, .350 6 .502 32, .200 1, .00 20 .03 A C
ATOM 2411 CD2 LEU A 326 6, .471 4 .338 33, .387 1, .00 17 .55 A C
ATOM 2412 C LEU A 326 2, .287 3 .605 31, ,982 1, .00 19 .08 A C
ATOM 2413 O LEU A 326 1, .589 3, .926 30, .989 1, .00 18 .42 A O
ATOM 2414 N SER A 327 1, .810 3 .326 33, .201 1, .00 19 .13 A N
ATOM 2415 CA SER A 327 0. .438 3 ,607 33, .672 1, .00 19, .58 A C
ATOM 2416 CB SER A 327 -0. .123 2, .391 34, .397 1, .00 19, .04 A C
ATOM 2417 OG SER A 327 -0. .176 1, .357 33. .434 1, .00 19, .38 A O
ATOM 2418 C SER A 327 0. .558 4 , .902 34. .476 1, .00 19, .58 A C
ATOM 2419 O SER A 327 1. ,609 5, .154 35. .075 1. .00 18, .88 A O
ATOM 2420 N THR A 328 -0. ,505 5, .696 34. ,595 1. .00 20, .38 A N
ATOM 2421 CA THR A 328 -0. ,861 6, ,454 35. .789 1. .00 19, .96 A C
ATOM 2422 CB THR A 328 -2, ,343 6, .678 35, .951 1, .00 20 .43 A C
ATOM 2423 OGl THR A 328 -2, .870 7, .047 34, .681 1, .00 20 .32 A O
ATOM 2424 CG2 THR A 328 -2, .602 7 .922 36, .842 1, .00 21 .84 A C
ATOM 2425 C THR A 328 -0. ,102 6, .262 37, .084 1, .00 19 .51 A C
ATOM 2426 O THR A 328 -0. ,223 5, .222 37, .739 1, .00 19, .59 A O
ATOM 2427 N SER A 329 0. .732 7, .268 37, .356 1, .00 18, .40 A N
ATOM 2428 CA SER A 329 1. .489 7. .464 38. .588 1. .00 19, .19 A C
ATOM 2429 CB : BSER A 329 0. ,679 7. .017 39. .818 0. .50 19, .34 A C
ATOM 2430 CB . ASER A 329 0. ,629 7, .141 39. .833 0, .50 19. .52 A C
ATOM 2431 OG : BSER A 329 0. .653 5, ,599 39. .887 0, .50 18. .73 A O
ATOM 2432 OG . ASER A 329 -0. ,672 7 , .722 39. .718 0. .50 20. ,52 A O
ATOM 2433 C SER A 329 2. ,792 6, .686 38. .588 1. .00 18. .71 A C
ATOM 2434 O SER A 329 3. ,533 6, .753 39. .550 1, .00 18. .11 A O
ATOM 2435 N GLN A 330 3. .066 5 , ,936 37. .524 1 , .00 17, .70 A N ATOM 2436 CA GLN A 330 4.339 5.250 37.420 1.00 17.71 A C
ATOM 2437 CB GLN A 330 4 .193 4 .001 36 .566 1 .00 17 .37 A C
ATOM 2438 CG GLN A 330 3 .233 2 .970 37 .168 1 .00 17 .60 A C
ATOM 2439 CD GLN A 330 3 .116 1 .700 36 .319 1, .00 18 .11 A C
ATOM 2440 OEl GLN A 330 3 .305 1 .763 35 .119 1 .00 16 .08 A 0
ATOM 2441 NE2 GLN A 330 2 .762 0 .550 36 .952 1 .00 15 .56 A N
ATOM 2442 C GLN A 330 5. .401 6 .195 36 .837 1, .00 18 .14 A C
ATOM 2443 O GLN A 330 5 .103 7 .308 36 .423 1, .00 18 .07 A 0
ATOM 2444 N LYS A 331 6 .643 5 .750 36 .842 1 .00 18 .66 A N
ATOM 2445 CA LYS A 331 7 .710 6 .524 36 .276 1 .00 19 .23 A C
ATOM 2446 CB LYS A 331 8 .229 7 .550 37 .289 1, .00 20 .19 A C
ATOM 2447 CG LYS A 331 8 .972 6 .934 38 .450 1 .00 23 .65 A C
ATOM 2448 CD LYS A 331 9 .071 7 .912 39 .625 1 .00 28 .70 A C
ATOM 2449 CE LYS A 331 9 .954 7 .332 40 .754 1 .00 31 .64 A C
ATOM 2450 NZ LYS A 331 10 .409 8 .388 41. .729 1 .00 34 .93 A N
ATOM 2451 C LYS A 331 8 .804 5 .589 35 .820 1 .00 18 .87 A C
ATOM 2452 O LYS A 331 8 .887 4 .410 36, .261 1 .00 18 .18 A 0
ATOM 2453 N ALA A 332 9 .587 6 .091 34 .873 1 .00 17 .37 A N
ATOM 2454 CA ALA A 332 10 .797 5 .406 34, .412 1 .00 17 .82 A C
ATOM 2455 CB ALA A 332 10, .689 5 .068 32, ,941 1, .00 17 .06 A C
ATOM 2456 C ALA A 332 11 .991 6 .324 34, .650 1 .00 17 .33 A C
ATOM 2457 O ALA A 332 11 .999 7 .480 34, .213 1 .00 16 .52 A 0
ATOM 2458 N THR A 333 13 .005 5 .805 35, .325 1 .00 17 .85 A N
ATOM 2459 CA THR A 333 14, .108 6 .643 35, .784 1 .00 17 .61 A C
ATOM 2460 CB THR A 333 14 .194 6 .544 37, .304 1 .00 18 .03 A C
ATOM 2461 OGl THR A 333 12. .956 6 .966 37 .902 1 .00 19 .93 A 0
ATOM 2462 CG2 THR A 333 15, .234 7 .490 37. ,851 1, .00 18 .18 A C
ATOM 2463 C THR A 333 15, .410 6 .186 35, .159 1 .00 17 .36 A C
ATOM 2464 O THR A 333 15 .727 4 .987 35. .162 1 .00 17 .40 A 0
ATOM 2465 N TYR A 334 16. .176 7, .135 34. ,628 1 .00 17 .05 A N
ATOM 2466 CA TYR A 334 17, .437 6, .840 33. ,986 1, .00 17, .42 A C
ATOM 2467 CB TYR A 334 17, .308 6 .975 32, .464 1, .00 16 .94 A C
ATOM 2468 CG TYR A 334 16 .144 6 .230 31, ,860 1 .00 16 .41 A C
ATOM 2469 CDl TYR A 334 16. .273 4, .891 31. .458 1, .00 14 .43 A C
ATOM 2470 CEl TYR A 334 15, .205 4 , .205 30. ,912 1, .00 14, .63 A C
ATOM 2471 CZ TYR A 334 13, .977 4 .846 30. .772 1, .00 15, .02 A C
ATOM 2472 OH TYR A 334 12, .929 4, ,188 30, ,216 1. .00 16, .23 A 0
ATOM 2473 CE2 TYR A 334 13. ,819 6. .147 31. .153 1, .00 15, .62 A c
ATOM 2474 CD2 TYR A 334 14. .907 6, ,835 31. ,718 1, .00 16, .76 A C
ATOM 2475 C TYR A 334 18. ,542 7, .767 34. ,455 1. ,00 17, .66 A C
ATOM 2476 O TYR A 334 18, .279 8, .840 34. .991 1. .00 16, .79 A 0
ATOM 2477 N SER A 335 19. .783 7, .375 34. .169 1, .00 17 .44 A N
ATOM 2478 CA SER A 335 20, .939 8, .218 34, ,442 1. .00 18, .62 A C
ATOM 2479 CB 1 BSER A 335 21. .879 7, .537 35. ,433 0. ,50 18, .54 A C
ATOM 2480 CB ; ASER A 335 21, .916 7 , ,518 35. ,393 0 , .50 18, .59 A C
ATOM 2481 OG ] BSER A 335 22. .697 6, .585 34. ,783 0 , .50 18 .97 A 0
ATOM 2482 OG , ASER A 335 21. ,316 7. ,174 36. 629 0. ,50 19. .43 A 0
ATOM 2483 C SER A 335 21. .680 8, ,538 33. ,128 1 , .00 18, .52 A C
ATOM 2484 O SER A 335 21, .698 7 .720 32. ,221 1, .00 18, .14 A 0
ATOM 2485 N PHE A 336 22. ,298 9. ,715 33. 049 1. ,00 17. .92 A N
ATOM 2486 CA PHE A 336 23. .115 10. .092 31. 911 1. ,00 18. .36 A C
ATOM 2487 CB PHE A 336 22, .324 10. .949 30. ,900 1 , .00 18, ,52 A C
ATOM 2488 CG PHE A 336 23, .150 11 .401 29. ,753 1, .00 17 .47 A C
ATOM 2489 CDl PHE A 336 23. ,733 12. .667 29. 739 1. ,00 18. .88 A c
ATOM 2490 CEl PHE A 336 24. ,529 13 , .067 28. 654 1. ,00 18, .14 A C
ATOM 2491 CZ PHE A 336 24, .749 12, .198 27. ,591 1 , .00 18. .35 A C
ATOM 2492 CE2 PHE A 336 24. ,174 10, ,936 27. ,601 1. ,00 18. .49 A C
ATOM 2493 CD2 PHE A 336 23. .386 10. .543 28. 681 1, ,00 19. ,03 A C
ATOM 2494 C PHE A 336 24. .314 10, ,898 32. ,403 1. .00 18, .74 A C
ATOM 2495 O PHE A 336 24. .159 11. .810 33. 195 1. ,00 18. ,98 A 0
ATOM 2496 N THR A 337 25, .504 10. .547 31. ,938 1 , .00 19. .28 A N
ATOM 2497 CA THR A 337 26. .733 11. .219 32. 364 1. ,00 19. ,77 A C
ATOM 2498 CB THR A 337 27. .879 10. .201 32. ,343 1, .00 20. ,11 A C ATOM 2499 OGl THR A 337 27.609 9.175 33.321 1.00 19.75 A O
ATOM 2500 CG2 THR A 337 29 .159 10 .857 32 .796 1 .00 21 .58 A C
ATOM 2501 C THR A 337 27 .096 12 .369 31 .440 1 .00 20 .14 A C
ATOM 2502 O THR A 337 27 .266 12 .163 30 .253 1 .00 20 .10 A O
ATOM 2503 N ALA A 338 27 .181 13 .571 32 .000 1 .00 19 .74 A N
ATOM 2504 CA ALA A 338 27 .487 14 .793 31 .259 1 .00 20 .23 A C
ATOM 2505 CB ALA A 338 26 .468 15 .858 31 .588 1 .00 19 .43 A C
ATOM 2506 C ALA A 338 28 .881 15 .292 31 .633 1 .00 20 .94 A C
ATOM 2507 O ALA A 338 29 .389 14 .991 32 .710 1 .00 19 .43 A O
ATOM 2508 N GLN A 339 29 .503 16 .042 30 .741 1 .00 22 .51 A N
ATOM 2509 CA GLN A 339 30 .750 16 .711 31 .070 1 .00 23 .12 A C
ATOM 2510 CB GLN A 339 31 .893 16 .162 30 .230 1 .00 24 .34 A C
ATOM 2511 CG GLN A 339 32 .591 14 .904 30 .726 1 .00 29 .11 A C
ATOM 2512 CD GLN A 339 34 .116 14 .923 30 .437 1 .00 36 .99 A C
ATOM 2513 OEl GLN A 339 34 .841 13 .991 30 .825 1 .00 41 .39 A O
ATOM 2514 NE2 GLN A 339 34 .597 15 .995 29 .778 1 .00 36 .90 A N
ATOM 2515 C GLN A 339 30 .543 18 .167 30 .722 1 .00 23 .27 A C
ATOM 2516 O GLN A 339 30 .034 18 .485 29 .641 1 .00 23 .02 A O
ATOM 2517 N ALA A 340 30 .922 19 .061 31 .619 1 .00 22 .60 A N
ATOM 2518 CA ALA A 340 30 .793 20 .492 31 .347 1 .00 22 .71 A C
ATOM 2519 CB ALA A 340 31 .311 21 .296 32 .535 1. .00 22 .84 A C
ATOM 2520 C ALA A 340 31 .524 20 .916 30 .076 1. .00 22 .30 A C
ATOM 2521 O ALA A 340 32 .474 20 .270 29 .650 1 .00 22 .44 A O
ATOM 2522 N GLY A 341 31 .063 21 .996 29 .455 1 .00 23 .38 A N
ATOM 2523 CA GLY A 341 31 .738 22 .554 28 .283 1 .00 23 .73 A C
ATOM 2524 C GLY A 341 30 .989 22 .425 26 .956 1 .00 24 .15 A C
ATOM 2525 O GLY A 341 31 .457 22 .902 25 .917 1. .00 24 .10 A O
ATOM 2526 N LYS A 342 29, .829 21 .774 26, .970 1. .00 24, .05 A N
ATOM 2527 CA LYS A 342 29, .038 21 .637 25, .743 1, .00 24. .39 A C
ATOM 2528 CB LYS A 342 29, .643 20 .545 24, .861 1, .00 25, .08 A C
ATOM 2529 CG LYS A 342 29, .610 19 .148 25, .496 1, .00 27, ,13 A C
ATOM 2530 CD LYS A 342 30, .471 18 .173 24, .723 1, ,00 29, .40 A C
ATOM 2531 CE LYS A 342 30, .254 16 .725 25. .182 1, .00 29, .96 A C
ATOM 2532 NZ LYS A 342 30, .738 16 .515 26. .576 1, .00 32, .09 A N
ATOM 2533 C LYS A 342 27, .552 21 .373 26, .058 1, .00 23, .40 A C
ATOM 2534 O LYS A 342 27, .220 20 .861 27, .144 1, .00 23, .54 A O
ATOM 2535 N PRO A 343 26, .652 21, .755 25, .151 1, .00 22, .09 A N
ATOM 2536 CA PRO A 343 25, .219 21, .683 25, ,450 1, .00 21, .01 A C
ATOM 2537 CB PRO A 343 24, .557 22, ,206 24, .163 1, .00 21, .58 A C
ATOM 2538 CG PRO A 343 25, .613 23, .026 23, .492 1, .00 22 , .06 A C
ATOM 2539 CD PRO A 343 26, .902 22, .348 23, ,820 1. .00 22 , .30 A C
ATOM 2540 C PRO A 343 24, ,729 20, .279 25, .756 1, .00 19, .35 A C
ATOM 2541 O PRO A 343 25, .311 19, .298 25, .317 1, .00 17. .82 A O
ATOM 2542 N LEU A 344 23, .645 20. .223 26, .521 1. .00 18, ,12 A N
ATOM 2543 CA LEU A 344 22, .945 18. .988 26, ,790 1, .00 17, .07 A C
ATOM 2544 CB LEU A 344 23, .019 18 , ,680 28, .278 1, .00 17, .12 A C
ATOM 2545 CG LEU A 344 22 , .250 17, ,476 28, .788 1. .00 16, .96 A C
ATOM 2546 CDl LEU A 344 22, .743 16, .188 28, ,128 1, .00 16, .29 A C
ATOM 2547 CD2 LEU A 344 22. .399 17, .414 30. .336 1. ,00 17. .28 A C
ATOM 2548 C LEU A 344 21. .484 19, .168 26. .360 1. .00 16, .41 A C
ATOM 2549 O LEU A 344 20. .814 20, .029 26. ,870 1. ,00 17. .49 A O
ATOM 2550 N LYS A 345 21. .013 18, .336 25. .440 1. ,00 15. .23 A N
ATOM 2551 CA LYS A 345 19. .638 18. .405 24. .943 1. ,00 14. ,51 A C
ATOM 2552 CB LYS A 345 19. ,644 18. .807 23. ,474 1. ,00 14. .84 A C
ATOM 2553 CG LYS A 345 20. .104 20. .235 23. .248 1. ,00 13. .82 A C
ATOM 2554 CD LYS A 345 19. ,987 20, .664 21. ,795 1. ,00 16. .32 A C
ATOM 2555 CE LYS A 345 18. ,599 21, .126 21. ,423 1. .00 13. .96 A C
ATOM 2556 NZ LYS A 345 18. ,513 21. .491 19. .992 1. .00 17. .70 A N
ATOM 2557 C LYS A 345 18. .929 17 , .066 25. .135 1. ,00 14. .25 A C
ATOM 2558 O LYS A 345 19. ,399 16 , ,033 24. .658 1. ,00 14. .33 A O
ATOM 2559 N ILE A 346 17. .821 17, .084 25. .870 1. ,00 13. .41 A N
ATOM 2560 CA ILE A 346 17, .031 15 .888 26, .116 1, .00 13, .30 A C
ATOM 2561 CB ILE A 346 16. .983 15 .589 27, .619 1, .00 13, .25 A C ATOM 2562 CGI ILE A 346 18.376 15.444 28.197 1.00 13.74 A C
ATOM 2563 CDl ILE A 346 18 .459 15 .807 29 .637 1 .00 15 .75 A C
ATOM 2564 CG2 ILE A 346 16 .180 14 .329 27 .874 1 .00 13 .54 A C
ATOM 2565 C ILE A 346 15 .598 16 .084 25 .601 1 .00 13 .26 A C
ATOM 2566 O ILE A 346 14 .900 17 .010 26 .020 1 .00 13 .15 A O
ATOM 2567 N SER A 347 15 .159 15 .197 24 .714 1 .00 12 .65 A N
ATOM 2568 CA SER A 347 13 .795 15 .262 24 .172 1 .00 12 .47 A C
ATOM 2569 CB : BSER A 347 13 .838 15 .473 22 .654 0 .35 12 .36 A C
ATOM 2570 CB . ASER A 347 13 .813 15 .524 22 .662 0 .65 12 .73 A C
ATOM 2571 OG : BSER A 347 12 .569 15 .297 22 .042 0 .35 10 .59 A O
ATOM 2572 OG , ASER A 347 14 .655 16 .634 22 .329 0 .65 13 .18 A O
ATOM 2573 C SER A 347 13 .032 13 .983 24 .491 1 .00 11 .72 A C
ATOM 2574 O SER A 347 13 .511 12 .881 24 .219 1 .00 11 .81 A O
ATOM 2575 N LEU A 348 11 .830 14 .165 25 .026 1 .00 11 .03 A N
ATOM 2576 CA LEU A 348 10 .864 13 .121 25 .289 1 .00 10 .93 A C
ATOM 2577 CB LEU A 348 10 .302 13 .274 26 .706 1 .00 10 .96 A C
ATOM 2578 CG LEU A 348 9 .054 12 .502 27 .097 1 .00 10 .80 A C
ATOM 2579 CDl LEU A 348 9 .396 11 .029 27 .180 1 .00 13 .05 A C
ATOM 2580 CD2 LEU A 348 8 .542 12 .969 28 .443 1. .00 12 .50 A C
ATOM 2581 C LEU A 348 9 .735 13 .231 24 .287 1 .00 11 .09 A C
ATOM 2582 O LEU A 348 9 .152 14 .302 24, .140 1. .00 12 .01 A O
ATOM 2583 N VAL A 349 9 .389 12 .127 23, .631 1 .00 10 .58 A N
ATOM 2584 CA VAL A 349 8 .327 12 .142 22, .638 1 .00 11 .32 A C
ATOM 2585 CB VAL A 349 8 .876 12 .223 21, .185 1 .00 11 .27 A C
ATOM 2586 CGI VAL A 349 7, .745 12 .102 20, .169 1 .00 12 .19 A C
ATOM 2587 CG2 VAL A 349 9. .653 13 .511 20, .961 1 .00 11 .93 A C
ATOM 2588 C VAL A 349 7 .522 10 .873 22 .768 1 .00 11 .68 A C
ATOM 2589 O VAL A 349 8 .099 9 .802 22 .870 1 .00 12 .43 A O
ATOM 2590 N TRP A 350 6 .200 10 .993 22 .768 1. .00 11 .69 A N
ATOM 2591 CA TRP A 350 5 .354 9 .813 22, .662 1 .00 11 .48 A C
ATOM 2592 CB TRP A 350 4 .719 9 .442 24, .002 1 .00 11 .79 A C
ATOM 2593 CG TRP A 350 3, .822 10 .448 24, .628 1 .00 11 .11 A C
ATOM 2594 CDl TRP A 350 2, .457 10, .378 24, .720 1, .00 12 .11 A C
ATOM 2595 NEl TRP A 350 1, .961 11, .469 25, .386 1, .00 12, .24 A N
ATOM 2596 CE2 TRP A 350 3, .015 12, .262 25, .774 1, .00 13 .16 A C
ATOM 2597 CD2 TRP A 350 4, ,208 11, .640 25. .311 1, .00 13, .33 A C
ATOM 2598 CE3 TRP A 350 5, .440 12 , .249 25. .593 1, .00 12, .51 A C
ATOM 2599 CZ3 TRP A 350 5, .444 13 , .449 26. .311 1, .00 13, .31 A C
ATOM 2600 CH2 TRP A 350 4, .248 14, .022 26. ,767 1, .00 13, .75 A C
ATOM 2601 CZ2 TRP A 350 3 , ,022 13 , .427 26. ,507 1, .00 13, .59 A C
ATOM 2602 C TRP A 350 4, .314 9 , .883 21. .536 1, .00 11, ,50 A C
ATOM 2603 O TRP A 350 3. .905 10, .953 21. .077 1, .00 11, .90 A O
ATOM 2604 N SER A 351 3, .921 8, .707 21. .071 1, .00 11, .83 A N
ATOM 2605 CA SER A 351 2, .889 8, .607 20. .070 1, .00 12, .20 A C
ATOM 2606 CB SER A 351 3, .182 7, ,496 19. .070 1, .00 12, .11 A C
ATOM 2607 OG SER A 351 4, ,356 7 , .772 18. ,310 1 , ,00 11, .85 A O
ATOM 2608 C SER A 351 1, .636 8, .378 20. .884 1 , .00 12 , .65 A C
ATOM 2609 O SER A 351 1. .360 7 , .285 21. ,375 1. .00 12, .72 A O
ATOM 2610 N ASP A 352 0, ,947 9, .477 21, ,115 1. .00 13, .75 A N
ATOM 2611 CA ASP A 352 -0, .205 9 .532 21. .982 1, .00 14 .48 A C
ATOM 2612 CB ASP A 352 -0, .508 11 .003 22, .225 1, .00 14 .84 A C
ATOM 2613 CG ASP A 352 -1, .480 11 .251 23, .385 1, .00 16 .70 A C
ATOM 2614 ODl ASP A 352 -1, .655 10 .366 24, .260 1, .00 15 .50 A O
ATOM 2615 OD2 ASP A 352 -2, .115 12 .329 23, .458 1, .00 15 .19 A O
ATOM 2616 C ASP A 352 -1, .427 8 .842 21, .389 1, .00 15 .19 A C
ATOM 2617 O ASP A 352 -1, .569 8, .678 20, .155 1, .00 15 .52 A O
ATOM 2618 N ALA A 353 -2, .331 8, .434 22, ,273 1, .00 15 .26 A N
ATOM 2619 CA ALA A 353 -3. .689 8, .074 21. .853 1, .00 15 .65 A C
ATOM 2620 CB ALA A 353 -4, .526 7, .801 23, ,051 1, .00 15 .59 A C
ATOM 2621 C ALA A 353 -4, .325 9, .197 21, .018 1, .00 15 .42 A C
ATOM 2622 O ALA A 353 -4, .076 10, .374 21, .264 1, .00 15 .11 A O
ATOM 2623 N PRO A 354 -5, .157 8, .840 20, .041 1, .00 16 .50 A N
ATOM 2624 CA PRO A 354 -5, .858 9, .841 19, .235 1, .00 16 .92 A C ATOM 2625 CB PRO A 354 -6.724 9.003 18.287 1.00 17.36 A C
ATOM 2626 CG PRO A 354 -6 .790 7 .646 18 .897 1 .00 17 .45 A C
ATOM 2627 CD PRO A 354 -5. .499 7 .456 19 .640 1 .00 16 .81 A C
ATOM 2628 C PRO A 354 -6 .723 10 .771 20 .073 1 .00 18 .05 A C
ATOM 2629 O PRO A 354 -7 .420 10 .293 20 .957 1 .00 17 .51 A O
ATOM 2630 N GLY A 355 -6 .629 12 .074 19 .819 1 .00 18 .60 A N
ATOM 2631 CA GLY A 355 -7 .392 13 .071 20, .527 1 .00 20 .06 A C
ATOM 2632 C GLY A 355 -8 .773 13 .285 19 .936 1 .00 21 .21 A C
ATOM 2633 O GLY A 355 -9 .095 12 .758 18 .880 1 .00 22 .41 A O
ATOM 2634 N SER A 356 -9 .598 14 .050 20 .628 1 .00 22 .87 A N
ATOM 2635 CA SER A 356 -10 .939 14 .377 20, .145 1, .00 23 .97 A C
ATOM 2636 CB SER A 356 -11 .924 14 .555 21 .319 1 .00 24 .97 A C
ATOM 2637 OG SER A 356 -12 .771 15 .696 21 .117 1 .00 26 .85 A O
ATOM 2638 C SER A 356 -10 .901 15 .654 19 .320 1 .00 24 .04 A C
ATOM 2639 O SER A 356 -10 .151 16 .583 19 .635 1 .00 23 .54 A O
ATOM 2640 N THR A 357 -11 .714 15 .684 18, .261 1 .00 24 .61 A N
ATOM 2641 CA THR A 357 -11 .826 16 .846 17 .396 1 .00 25 .58 A C
ATOM 2642 CB THR A 357 -12 .423 16 .436 16 .032 1 .00 25 .86 A C
ATOM 2643 OGl THR A 357 -13 .673 15 .748 16, .218 1 .00 25 .91 A O
ATOM 2644 CG2 THR A 357 -11 .534 15 .392 15, .334 1, .00 25 .30 A C
ATOM 2645 C THR A 357 -12 .687 17 .982 18, .000 1 .00 26 .58 A C
ATOM 2646 O THR A 357 -12 .812 19 .035 17 .398 1 .00 26 .22 A O
ATOM 2647 N THR A 358 -13 .276 17 .771 19. .175 1 .00 27 .59 A N
ATOM 2648 CA THR A 358 -14 .113 18 .816 19. .779 1, .00 28 .37 A C
ATOM 2649 CB THR A 358 -15 .575 18 .335 19, .938 1 .00 28 .27 A C
ATOM 2650 OGl THR A 358 -15, .606 17 .065 20, .606 1 .00 28 .60 A O
ATOM 2651 CG2 THR A 358 -16 .192 18 .066 18, .587 1, .00 27 .97 A C
ATOM 2652 C THR A 358 -13 .605 19 .321 21, .118 1, .00 28 .71 A C
ATOM 2653 O THR A 358 -13 .954 20 .424 21, .524 1, .00 29 .22 A O
ATOM 2654 N ALA A 359 -12, .758 18, .548 21, .795 1 .00 28 .63 A N
ATOM 2655 CA ALA A 359 -12 .311 18 .925 23, ,133 1, .00 27 .94 A C
ATOM 2656 CB ALA A 359 -11 .668 17 .739 23, .814 1 .00 28 .64 A C
ATOM 2657 C ALA A 359 -11, .349 20 .100 23. .099 1, .00 27 .98 A C
ATOM 2658 O ALA A 359 -10, .738 20, .393 22, .060 1, .00 27 .61 A O
ATOM 2659 N SER A 360 -11, .213 20 .785 24, .241 1, .00 27 .18 A N
ATOM 2660 CA SER A 360 -10 .301 21 .916 24. .344 1 .00 27 .00 A C
ATOM 2661 CB SER A 360 -10, .351 22. .564 25. .737 1, .00 27 .57 A C
ATOM 2662 OG SER A 360 -11, .688 22, .840 26. ,125 1, .00 31, .54 A O
ATOM 2663 C SER A 360 -8, .858 21, .485 24. .060 1, .00 25 .14 A C
ATOM 2664 O SER A 360 -8, .115 22 .230 23. .446 1 .00 24 .27 A O
ATOM 2665 N LEU A 361 -8, ,478 20 .307 24. .553 1, .00 23 .74 A N
ATOM 2666 CA LEU A 361 -7. .100 19, .812 24. ,453 1 , .00 23, .54 A C
ATOM 2667 CB LEU A 361 -6, .480 19, .583 25. .840 1, .00 23 .87 A C
ATOM 2668 CG LEU A 361 -6, .119 20 .802 26. .702 1 .00 27 .30 A C
ATOM 2669 CDl LEU A 361 -5, .434 20, .335 27. .980 1, .00 28 .61 A C
ATOM 2670 CD2 LEU A 361 -5. .217 21, .827 25. ,975 1 , .00 28, .84 A C
ATOM 2671 C LEU A 361 -7, .097 18, .493 23. ,701 1, ,00 21 .78 A C
ATOM 2672 O LEU A 361 -7, .942 17 .611 23. .961 1, .00 21 .73 A O
ATOM 2673 N THR A 362 -6, .141 18, .325 22, ,790 1, .00 19 .81 A N
ATOM 2674 CA THR A 362 -6. ,053 17, .058 22, ,066 1. ,00 18, ,76 A C
ATOM 2675 CB THR A 362 -5, ,433 17, .230 20. ,657 1, .00 19 .42 A C
ATOM 2676 OGl THR A 362 -4, .100 17, .707 20. .786 1, .00 17 .51 A O
ATOM 2677 CG2 THR A 362 -6, .174 18, .305 19, .862 1 .00 20 .58 A C
ATOM 2678 C THR A 362 -5. ,261 16, .023 22. .819 1. .00 17, .21 A C
ATOM 2679 O THR A 362 -5, .411 14 , .858 22. .530 1 , .00 13 .79 A O
ATOM 2680 N LEU A 363 -4, .398 16, .448 23. .761 1, .00 16 ,68 A N
ATOM 2681 CA LEU A 363 -3, .560 15, .505 24. .484 1, .00 16 .39 A C
ATOM 2682 CB LEU A 363 -2. ,547 16, .213 25. .411 1. .00 16, .30 A C
ATOM 2683 CG LEU A 363 -1, .460 15. .318 25. ,990 1, ,00 16, .62 A C
ATOM 2684 CDl LEU A 363 -0, .380 14. .960 24. .939 1. .00 16 .31 A C
ATOM 2685 CD2 LEU A 363 -0, .838 15, .936 27. ,236 1. .00 15, .84 A C
ATOM 2686 C LEU A 363 -4. ,424 14, .536 25. ,280 1. ,00 17 , .11 A C
ATOM 2687 O LEU A 363 -5, ,404 14, .936 25. ,911 1, ,00 17, .15 A O ATOM 2688 N VAL A 364 -4.068 13.253 25.249 1.00 16.50 A N
ATOM 2689 CA VAL A 364 -4 .829 12 .263 25 .975 1 .00 16 .09 A C
ATOM 2690 CB VAL A 364 -5, .285 11 .121 25 .030 1 .00 15 .79 A C
ATOM 2691 CGI VAL A 364 -5, .871 9 .933 25 .826 1. .00 17 .24 A C
ATOM 2692 CG2 VAL A 364 -6 .288 11 .651 24. .020 1, .00 16 .26 A C
ATOM 2693 C VAL A 364 -3 .983 11 .744 27, .139 1, .00 15 .68 A C
ATOM 2694 O VAL A 364 -4 .329 11 .942 28, .309 1, .00 15 .17 A O
ATOM 2695 N ASN A 365 -2 .875 11 .085 26, .809 1, .00 14 .41 A N
ATOM 2696 CA ASN A 365 -1 .931 10 .614 27, .804 1, .00 14 .31 . A C
ATOM 2697 CB ASN A 365 -1 .302 9 .286 27, .354 1, .00 14 .31 A C
ATOM 2698 CG ASN A 365 -2 .342 8 .161 27, .214 1, .00 16 .21 A C
ATOM 2699 ODl ASN A 365 -3 .298 8 .081 28, .004 1, .00 13 .31 A O
ATOM 2700 ND2 ASN A 365 -2 .158 7 .283 26, .206 1, .00 14 .49 A N
ATOM 2701 C ASN A 365 -0, .858 11 .690 28, .088 1, .00 14 .80 A C
ATOM 2702 O ASN A 365 -0 .174 12 .190 27 .172 1 .00 14 .01 A O
ATOM 2703 N ASP A 366 -0 .696 12 .015 29 .360 1 .00 14 .06 A N
ATOM 2704 CA ASP A 366 0 .158 13 .115 29 .783 1 .00 15 .00 A C
ATOM 2705 CB ASP A 366 -0 .632 14 .068 30 .672 1 .00 14 .40 A C
ATOM 2706 CG ASP A 366 0 .105 15 .346 30 .990 1 .00 15 .13 A C
ATOM 2707 ODl ASP A 366 1 .344 15 .467 30 .710 1 .00 13 .15 A O
ATOM 2708 OD2 ASP A 366 -0 .491 16 .284 31 .609 1 .00 17 .79 A O
ATOM 2709 C ASP A 366 1 .367 12 .568 30 .548 1 .00 14 .89 A C
ATOM 2710 O ASP A 366 1 .257 12 .132 31 .708 1 .00 15 .57 A O
ATOM 2711 N LEU A 367 2 .501 12 .562 29 .865 1 .00 14 .16 A N
ATOM 2712 CA LEU A 367 3 .772 12 .248 30 .462 1 .00 14 .65 A C
ATOM 2713 CB LEU A 367 4 .581 11 .333 29 .520 1, .00 14 .37 A C
ATOM 2714 CG LEU A 367 3 .990 10 .005 29 .077 1, .00 13 .06 A c
ATOM 2715 CDl LEU A 367 5 .063 9 .220 28, .244 1, .00 12 .20 A C
ATOM 2716 CD2 LEU A 367 3, .485 9 .123 30, .239 1, .00 15 .25 A C
ATOM 2717 C LEU A 367 4, .523 13, .555 30, .710 1, .00 14 .15 A C
ATOM 2718 O LEU A 367 4, .271 14 .546 30, .045 1, .00 14 .17 A 0
ATOM 2719 N ASP A 368 5, .441 13 .556 31, .677 1, ,00 14 .04 A N
ATOM 2720 CA ASP A 368 6, .271 14, .705 31, .980 1, .00 14 .28 A C
ATOM 2721 CB ASP A 368 5, .959 15, .284 33, .354 1, .00 15 .52 A C
ATOM 2722 CG ASP A 368 4, .529 15, .726 33, .515 1, .00 17 .54 A C
ATOM 2723 ODl ASP A 368 3, .909 16. .253 32, .540 1, .00 14 .48 A O
ATOM 2724 OD2 ASP A 368 4 , .006 15. .624 34, .642 1, .00 17 .39 A O
ATOM 2725 C ASP A 368 7, .724 14, .275 32, .057 1, .00 14 .49 A C
ATOM 2726 O ASP A 368 8, .034 13, .203 32, .587 1, .00 13 .94 A O
ATOM 2727 N LEU A 369 8, .603 15, .108 31, .520 1. .00 14 .32 A N
ATOM 2728 CA LEU A 369 10, .044 14 , .937 31, .645 1 , .00 14 .31 A C
ATOM 2729 CB LEU A 369 10, .735 15, .448 30, .380 1, .00 13 .72 A C
ATOM 2730 CG LEU A 369 12, .238 15 .284 30, .298 1, .00 12 .96 A C
ATOM 2731 CDl LEU A 369 12, .572 13, .850 30, .345 1, .00 12 .52 A C
ATOM 2732 CD2 LEU A 369 12, .749 15, .953 28. .980 1, .00 14 .11 A C
ATOM 2733 C LEU A 369 10, .539 15, .733 32, .854 1, .00 15 .02 A C
ATOM 2734 O LEU A 369 10, .218 16, .922 33, .012 1. .00 15 .50 A 0
ATOM 2735 N VAL A 370 11, .315 15, .085 33, .698 1. .00 15 .26 A N
ATOM 2736 CA VAL A 370 11, .875 15, .729 34, .905 1. .00 15 .63 A C
ATOM 2737 CB VAL A 370 11, .144 15, .278 36, .180 1. .00 15 .95 A C
ATOM 2738 CGI VAL A 370 11, ,679 16, .020 37, .425 1. .00 17 .73 A C
ATOM 2739 CG2 VAL A 370 9, .687 15, .487 36, .024 1. .00 15 .57 A C
ATOM 2740 C VAL A 370 13, .359 15, .388 34, .975 1. .00 15 .46 A C
ATOM 2741 O VAL A 370 13, .767 14 , .219 35, .042 1. ,00 15, .96 A O
ATOM 2742 N ILE A 371 14, .174 16, ,422 34, .908 1. ,00 15 .15 A N
ATOM 2743 CA ILE A 371 15, .608 16. .261 34, .858 1. .00 14, .98 A C
ATOM 2744 CB ILE A 371 16, .171 17, .036 33, .669 1. ,00 14 , .71 A C
ATOM 2745 CGI ILE A 371 15, .509 16, .589 32, .336 1. ,00 14 , .63 A C
ATOM 2746 CDl ILE A 371 15, .600 15, .075 32, .072 1. ,00 14, .66 A C
ATOM 2747 CG2 ILE A 371 17, ,674 16. .922 33, .614 1. .00 12 , .85 A C
ATOM 2748 C ILE A 371 16, .145 16. .835 36. ,155 1. ,00 15, .95 A C
ATOM 2749 O ILE A 371 15 .648 17 .853 36 .618 1, ,00 16 .74 A O
ATOM 2750 N THR A 372 17 .150 16 .174 36 .727 1, .00 16 .86 A N ATOM 2751 CA THR A 372 17,.858 16.671 37,.897 1.00 17.19 A C
ATOM 2752 CB THR A 372 17, .618 15 .748 39, .089 1, .00 17 .36 A C
ATOM 2753 OGl THR A 372 16, .212 15 .514 39, .265 1 .00 18 .17 A O
ATOM 2754 CG2 THR A 372 18, .044 16 .409 40, .372 1, .00 17 .84 A C
ATOM 2755 C THR A 372 19, .364 16 .729 37, .634 1, .00 17 .65 A C
ATOM 2756 O THR A 372 19, .962 15 .725 37, .262 1 .00 18 .44 A O
ATOM 2757 N ALA A 373 19 .971 17 .891 37, .870 1 .00 17 .80 A N
ATOM 2758 CA ALA A 373 21 .376 18 .118 37, .606 1 .00 18 .13 A C
ATOM 2759 CB ALA A 373 21 .643 19 .607 37, .475 1 .00 18 .43 A C
ATOM 2760 C ALA A 373 22 .175 17 .545 38, .767 1 .00 19 .16 A C
ATOM 2761 O ALA A 373 21 .601 17 .188 39, .780 1 .00 18 .57 A O
ATOM 2762 N PRO A 374 23, .479 17 .368 38, .581 1 .00 19 .84 A N
ATOM 2763 CA PRO A 374 24, .348 16 .857 39, .642 1 .00 20 .99 A C
ATOM 2764 CB PRO A 374 25, .727 16 .884 39, .001 1, .00 20 .60 A C
ATOM 2765 CG PRO A 374 25, .434 16 .700 37, .530 1, .00 20 .96 A C
ATOM 2766 CD PRO A 374 24, .174 17 .460 37, .286 1, .00 20 .29 A C
ATOM 2767 C PRO A 374 24 .266 17 .647 40, .948 1 .00 22 .06 A C
ATOM 2768 O PRO A 374 24, .303 17 .039 42, .011 1, .00 23 .95 A O
ATOM 2769 N ASN A 375 24, .024 18 .954 40, .873 1, .00 23 .03 A N
ATOM 2770 CA ASN A 375 23, .910 19 .770 42, .058 1, .00 23 .32 A C
ATOM 2771 CB ASN A 375 24 .515 21 .165 41, .790 1, .00 24 .03 A C
ATOM 2772 CG ASN A 375 23 .581 22 .096 40, .993 1 .00 26 .93 A C
ATOM 2773 ODl ASN A 375 22 .515 21 .689 40 .492 1 .00 28 .51 A O
ATOM 2774 ND2 ASN A 375 23 .987 23 .362 40 .878 1 .00 27 .28 A N
ATOM 2775 C ASN A 375 22 .471 19 .898 42 .563 1. .00 23 .07 A C
ATOM 2776 O ASN A 375 22, .208 20 .711 43, .430 1 .00 22 .92 A O
ATOM 2777 N GLY A 376 21. .541 19 .120 42, .010 1. .00 21 .76 A N
ATOM 2778 CA GLY A 376 20. .166 19 .165 42, .469 1. .00 21 .43 A C
ATOM 2779 C GLY A 376 19. .197 20 .077 41, .724 1, .00 20 .91 A C
ATOM 2780 O GLY A 376 17, .990 20 .006 41, .937 1. .00 19 .94 A O
ATOM 2781 N THR A 377 19, .696 20 .909 40, .828 1, .00 21 .29 A N
ATOM 2782 CA THR A 377 18, .793 21 .785 40, .090 1, .00 21 .36 A C
ATOM 2783 CB THR A 377 19, .571 22 .738 39, .220 1. .00 21 .19 A C
ATOM 2784 OGl THR A 377 20, .423 23 .532 40, .054 1. .00 22 .26 A O
ATOM 2785 CG2 THR A 377 18, .635 23 .724 38, .538 1, .00 20 .97 A C
ATOM 2786 C THR A 377 17, .818 20 .971 39, .239 1. .00 20 .56 A C
ATOM 2787 O THR A 377 18, .206 20 .058 38, .541 1. .00 19 .93 A O
ATOM 2788 N LYS A 378 16, .558 21 .345 39, .315 1, .00 20 .68 A N
ATOM 2789 CA LYS A 378 15, .488 20 .630 38, .644 1. .00 21 .64 A C
ATOM 2790 CB LYS A 378 14, .321 20 .503 39, .594 1, .00 22 .42 A C
ATOM 2791 CG LYS A 378 13, .709 19 .168 39, .611 1, ,00 28 .13 A C
ATOM 2792 CD LYS A 378 14, .144 18 .449 40, .913 1, .00 33 .32 A C
ATOM 2793 CE LYS A 378 13, .743 17 .001 40, .854 1, .00 34 .92 A C
ATOM 2794 NZ LYS A 378 14, .605 16 .111 41, .699 1, .00 38 .61 A N
ATOM 2795 C LYS A 378 14, .990 21 .344 37, .397 1. .00 20 .06 A C
ATOM 2796 O LYS A 378 14 .902 22 .553 37, .388 1. .00 19 .59 A O
ATOM 2797 N TYR A 379 14, .623 20 .568 36, .378 1, ,00 18 .59 A N
ATOM 2798 CA TYR A 379 14, .009 21 .088 35, .155 1, .00 17 .87 A C
ATOM 2799 CB TYR A 379 15, .030 21 .111 34, .011 1. .00 17 .25 A C
ATOM 2800 CG TYR A 379 16 .382 21 .654 34, .386 1, .00 17 .53 A C
ATOM 2801 CDl TYR A 379 17, .297 20 .864 35, .052 1, ,00 18 .58 A C
ATOM 2802 CEl TYR A 379 18 .537 21 .352 35, .417 1, .00 19 .05 A C
ATOM 2803 CZ TYR A 379 18 .895 22 .655 35, .097 1, .00 21 .25 A C
ATOM 2804 OH TYR A 379 20 .160 23 .104 35, .465 1, .00 20 .46 A O
ATOM 2805 CE2 TYR A 379 18, .004 23 .459 34, .399 1, .00 19 .58 A C
ATOM 2806 CD2 TYR A 379 16, .751 22 .953 34, .060 1, ,00 18 .07 A C
ATOM 2807 C TYR A 379 12 .852 20 .198 34, .732 1, .00 17 .42 A C
ATOM 2808 O TYR A 379 12 .967 18 .973 34, .766 1, .00 18 .13 A O
ATOM 2809 N VAL A 380 11 .732 20 .787 34 .340 1, .00 16 .44 A N
ATOM 2810 CA VAL A 380 10 .653 19 .989 33 .750 1, .00 16 .11 A C
ATOM 2811 CB VAL A 380 9 .320 20 .114 34 .514 1, .00 16 .66 A C
ATOM 2812 CGI VAL A 380 9 .521 19 .742 36 .000 1, .00 17 .64 A C
ATOM 2813 CG2 VAL A 380 8 .716 21 .505 34 .369 1, .00 16 .52 A C ATOM 2814 C VAL A 380 10,.466 20.353 32..283 1.00 14.93 A C
ATOM 2815 O VAL A 380 10, .876 21 .425 31. .826 1 .00 15 .17 A O
ATOM 2816 N GLY A 381 9, .868 19 .436 31, .547 1 .00 14 .19 A N
ATOM 2817 CA GLY A 381 9, .761 19 .541 30. .101 1 .00 13 .85 A C
ATOM 2818 C GLY A 381 9, .132 20 .847 29, .647 1 .00 13 .74 A C
ATOM 2819 O GLY A 381 8, .096 21 .259 30, .153 1 .00 13 .57 A O
ATOM 2820 N ASN A 382 9, .813 21 .509 28, .729 1 .00 13 .87 A N
ATOM 2821 CA ASN A 382 9 .332 22 .719 28, .064 1 .00 14 .08 A C
ATOM 2822 CB ASN A 382 8 .002 22 .451 27, .344 1 .00 13 .91 A C
ATOM 2823 CG ASN A 382 8 .148 21 .436 26, .209 1 .00 14 .03 A C
ATOM 2824 ODl ASN A 382 9 .250 21 .181 25, .757 1 .00 13 .23 A O
ATOM 2825 ND2 ASN A 382 7 .041 20 .850 25. .770 1 .00 10 .67 A N
ATOM 2826 C ASN A 382 9 .232 23 .942 28. .966 1 .00 14 .58 A C
ATOM 2827 O ASN A 382 8 .682 24 .981 28, .556 1 .00 14 .15 A O
ATOM 2828 N ASP A 383 9 .796 23 .869 30, .178 1 .00 14 .75 A N
ATOM 2829 CA ASP A 383 9, .813 25 .057 31, .017 1 .00 14 .97 A C
ATOM 2830 CB ASP A 383 9, .593 24 .709 32. ,499 1 .00 15 .34 A C
ATOM 2831 CG ASP A 383 9 .580 25 .952 33, .388 1 .00 15 .81 A C
ATOM 2832 ODl ASP A 383 9, .786 27 .082 32. .845 1 .00 16 .83 A O
ATOM 2833 OD2 ASP A 383 9, .394 25 .897 34. .636 1 .00 15 .84 A O
ATOM 2834 C ASP A 383 11 .127 25 .813 30. ,810 1 .00 15 .13 A C
ATOM 2835 O ASP A 383 12, .160 25 .490 31. .398 1 .00 15 .46 A 0
ATOM 2836 N PHE A 384 11, .074 26 .859 30. .000 1 .00 15 .11 A N
ATOM 2837 CA PHE A 384 12, .284 27 .559 29. ,589 1 .00 15 .10 A C
ATOM 2838 CB PHE A 384 12, .178 27 .818 28. .086 1 .00 16 .52 A C
ATOM 2839 CG PHE A 384 12, .247 26 .560 27. ,240 1. .00 14 .23 A C
ATOM 2840 CDl PHE A 384 13, .440 25 .910 27. ,059 1. .00 19 .41 A C
ATOM 2841 CEl PHE A 384 13, .516 24 .782 26. .273 1, .00 19 .08 A C
ATOM 2842 CZ PHE A 384 12, .395 24 .303 25. .685 1, .00 18 .05 A C
ATOM 2843 CE2 PHE A 384 11, ,208 24 .943 25. ,845 1, .00 14 .73 A C
ATOM 2844 CD2 PHE A 384 11, .140 26 .070 26. ,602 1, .00 16 .74 A C
ATOM 2845 C PHE A 384 12, .546 28 .857 30. .389 1 .00 15 .96 A c
ATOM 2846 O PHE A 384 13 .547 29 .558 30. ,152 1, .00 15 .13 A 0
ATOM 2847 N THR A 385 11, .666 29 .151 31. ,350 1, .00 16 .18 A N
ATOM 2848 CA THR A 385 11, .820 30 .294 32. .264 1 .00 17 .37 A c
ATOM 2849 CB THR A 385 10 .519 31 .097 32. ,400 1 .00 17 .19 A c
ATOM 2850 OGl THR A 385 9, .520 30 .295 33. .030 1 .00 17 .98 A O
ATOM 2851 CG2 THR A 385 9, .922 31 .491 31. .028 1 .00 18 .21 A c
ATOM 2852 C THR A 385 12, .238 29 .868 33. .689 1 .00 17 .95 A c
ATOM 2853 O THR A 385 11, .703 28 .890 34. .252 1 .00 17 .41 A 0
ATOM 2854 N ALA A 386 13, ,197 30 .599 34. .250 1 .00 18 .54 A N
ATOM 2855 CA ALA A 386 13, .743 30 .266 35. .565 1 .00 19 .43 A c
ATOM 2856 CB ALA A 386 15, .056 30 .971 35. .792 1 .00 19 .87 A C
ATOM 2857 C ALA A 386 12, .728 30 .697 36. .594 1 .00 20 .06 A C
ATOM 2858 O ALA A 386 12, .078 31 .735 36. .409 1 .00 21 .03 A O
ATOM 2859 N PRO A 387 12, .525 29 .897 37. .635 1 .00 19 .71 A N
ATOM 2860 CA PRO A 387 13, .134 28 .582 37. .766 1 .00 20 .17 A C
ATOM 2861 CB PRO A 387 12, .951 28 .269 39. .250 1 .00 20 .90 A C
ATOM 2862 CG PRO A 387 11, .609 28 .910 39. ,587 1 .00 20 .16 A C
ATOM 2863 CD PRO A 387 11, .670 30 .223 38. .796 1 .00 21 .11 A C
ATOM 2864 C PRO A 387 12, .413 27 .549 36. .890 1 .00 19 .33 A C
ATOM 2865 O PRO A 387 11, .237 27 .688 36. .612 1 .00 18 .07 A O
ATOM 2866 N TYR A 388 13, .144 26 .521 36. .491 1 .00 19 .24 A N
ATOM 2867 CA TYR A 388 12, .781 25 .672 35. .365 1 .00 19 .24 A C
ATOM 2868 CB TYR A 388 14, .059 25 .211 34. .661 1 .00 18 .91 A C
ATOM 2869 CG TYR A 388 14, .912 26 .363 34. .177 1 .00 18 .51 A C
ATOM 2870 CDl TYR A 388 16, .128 26 .625 34. .761 1, .00 16 .61 A C
ATOM 2871 CEl TYR A 388 16, .912 27 .701 34. .350 1 .00 17 .76 A C
ATOM 2872 CZ TYR A 388 16 .462 28 .511 33. .312 1 .00 15 .12 A C
ATOM 2873 OH TYR A 388 17 .242 29 .565 32. .918 1 .00 17 .69 A O
ATOM 2874 CE2 TYR A 388 15 .241 28 .276 32, .723 1 .00 14 .50 A c
ATOM 2875 CD2 TYR A 388 14 .462 27 .229 33. .154 1 .00 14 .33 A c
ATOM 2876 C TYR A 388 11 .934 24 .467 35. .745 1 .00 19 .73 A c ATOM 2877 O TYR A 388 11.688 23.598 34.913 1.00 19.79 A O
ATOM 2878 N ASP A 389 11 .422 24 .442 36 .972 1 .00 20 .38 A N
ATOM 2879 CA ASP A 389 10 .605 23 .327 37 .430 1 .00 21 .41 A C
ATOM 2880 CB ASP A 389 11 .346 22 .547 38 .507 1 .00 22 .04 A C
ATOM 2881 CG ASP A 389 11 .504 23 .343 39 .796 1 .00 24 .83 A C
ATOM 2882 ODl ASP A 389 11 .618 22 .706 40 .869 1 .00 27 .95 A O
ATOM 2883 OD2 ASP A 389 11 .523 24 .595 39 .822 1 .00 24 .63 A O
ATOM 2884 C ASP A 389 9 .246 23 .724 37 .968 1 .00 21 .64 A C
ATOM 2885 O ASP A 389 8. .629 22 .947 38 .709 1 .00 22 .12 A O
ATOM 2886 N ASN A 390 8 .759 24 .908 37, .618 1 .00 21 .45 A N
ATOM 2887 CA ASN A 390 7 .455 25 .326 38, .130 1 .00 22 .14 A C
ATOM 2888 CB ASN A 390 7 .555 26 .664 38, .892 1 .00 22 .91 A C
ATOM 2889 CG ASN A 390 7 .965 27 .825 37, .989 1 .00 23 .08 A C
ATOM 2890 ODl ASN A 390 8 .404 27 .620 36, .847 1 .00 22 .98 A O
ATOM 2891 ND2 ASN A 390 7 .816 29 .050 38 .491 1 .00 23 .21 A N
ATOM 2892 C ASN A 390 6 .356 25 .402 37 .060 1 .00 22 .16 A C
ATOM 2893 O ASN A 390 5 .181 25 .500 37 .405 1 .00 21 .88 A O
ATOM 2894 N ASN A 391 6 .717 25 .340 35 .772 1 .00 21 .51 A N
ATOM 2895 CA ASN A 391 5 .705 25 .411 34 .713 1 .00 20 .98 A C
ATOM 2896 CB ASN A 391 5 .986 26 .567 33 .728 1 .00 21 .22 A C
ATOM 2897 CG ASN A 391 6 .221 27 .924 34, .426 1. .00 22 .34 A C
ATOM 2898 ODl ASN A 391 7 .345 28 .481 34, .388 1 .00 22 .21 A O
ATOM 2899 ND2 ASN A 391 5 .151 28 .490 35, .029 1 .00 23 .08 A N
ATOM 2900 C ASN A 391 5 .611 24 .072 33, .978 1 .00 20 .79 A C
ATOM 2901 O ASN A 391 6 .295 23 .818 32, .978 1 .00 21 .08 A O
ATOM 2902 N TRP A 392 4 .741 23 .211 34, .467 1, .00 20 .10 A N
ATOM 2903 CA TRP A 392 4 .601 21 .862 33, ,928 1, .00 20 .04 A C
ATOM 2904 CB TRP A 392 3 .893 20 .979 34, .926 1, .00 20 .74 A C
ATOM 2905 CG TRP A 392 4 .629 20 .757 36, .231 1, .00 24 .30 A C
ATOM 2906 CDl TRP A 392 4 .687 21 .605 37. ,309 1, ,00 28 .54 A C
ATOM 2907 NEl TRP A 392 5, .435 21 .038 38. .317 1, .00 30. .71 A N
ATOM 2908 CE2 TRP A 392 5, .870 19 .804 37. ,902 1. .00 28, .29 A C
ATOM 2909 CD2 TRP A 392 5, .367 19 .594 36. .598 1, .00 27, .17 A C
ATOM 2910 CE3 TRP A 392 5. .695 18 .400 35. .937 1, .00 29, .38 A C
ATOM 2911 CZ3 TRP A 392 6, .456 17 .447 36. .608 1. .00 28, .54 A C
ATOM 2912 CH2 TRP A 392 6, .922 17 .683 37. .904 1, .00 30, .49 A C
ATOM 2913 CZ2 TRP A 392 6, .643 18 .857 38. .566 1. ,00 30, .31 A C
ATOM 2914 C TRP A 392 3. .767 21 .890 32. ,661 1. ,00 19, .03 A C
ATOM 2915 O TRP A 392 2, .828 22 .678 32. ,552 1. .00 19, .40 A O
ATOM 2916 N ASP A 393 4, .107 21 .020 31, ,709 1. .00 17, .66 A N
ATOM 2917 CA ASP A 393 3. .416 20 .958 30. ,424 1. ,00 16, .59 A C
ATOM 2918 CB ASP A 393 4, .431 20 .669 29. ,332 1. ,00 16, ,47 A C
ATOM 2919 CG ASP A 393 3, .813 20 .660 27. ,930 1. ,00 15. .80 A C
ATOM 2920 ODl ASP A 393 4 , .350 21 .364 27. ,045 1. ,00 14. .98 A O
ATOM 2921 OD2 ASP A 393 2, .817 19 .975 27. ,629 1. ,00 15. .12 A O
ATOM 2922 C ASP A 393 2, .324 19 .888 30. ,425 1. ,00 16. .46 A C
ATOM 2923 O ASP A 393 2, .606 18 .716 30. ,648 1. ,00 15. ,65 A O
ATOM 2924 N GLY A 394 1, .080 20 .302 30. ,178 1. ,00 15. .96 A N
ATOM 2925 CA GLY A 394 -0, .029 19 .384 30. ,014 1. .00 16. .13 A C
ATOM 2926 C GLY A 394 -0, ,747 19 .498 28. ,675 1. ,00 16. .27 A C
ATOM 2927 O GLY A 394 -1, .936 19 .255 28. .601 1. ,00 16, .15 A O
ATOM 2928 N ARG A 395 -0, .030 19 .864 27. ,617 1. ,00 17, .31 A N
ATOM 2929 CA ARG A 395 -0, ,607 19 .978 26. .264 1. ,00 17, .68 A C
ATOM 2930 CB ARG A 395 -0, .588 21 .437 25. .783 1. ,00 19, .08 A C
ATOM 2931 CG ARG A 395 -1, .434 22 .408 26. ,518 1. ,00 26, .02 A C
ATOM 2932 CD ARG A 395 -1, .172 23 .839 26. 066 1. ,00 31. .34 A C
ATOM 2933 NE ARG A 395 -1. .802 24, .813 26. ,969 1. ,00 37. .30 A N
ATOM 2934 CZ ARG A 395 -3, ,026 25, .331 26. 821 1. ,00 40. .58 A C
ATOM 2935 NHl ARG A 395 -3, ,478 26, .216 27. 717 1. ,00 44. .12 A N
ATOM 2936 NH2 ARG A 395 -3, ,805 24, .983 25. 805 1. 00 40. .20 A N
ATOM 2937 C ARG A 395 0, .176 19, .240 25. 165 1. 00 15. .80 A C
ATOM 2938 O ARG A 395 -0 .418 18 .827 24. ,176 1. .00 16, .01 A O
ATOM 2939 N ASN A 396 1 .502 19 .212 25. ,282 1. .00 14, .41 A N ATOM 2940 CA ASN A 396 2.389 18.645 24.251 1.00 13.89 A C
ATOM 2941 CB ASN A 396 3 .662 19 .483 24 .133 1 .00 13 .10 A C
ATOM 2942 CG ASN A 396 3. .408 20 .889 23 .585 1 .00 14 .63 A C
ATOM 2943 ODl ASN A 396 3 .129 21 .075 22 .374 1 .00 11 .53 A O
ATOM 2944 ND2 ASN A 396 3 .550 21 .897 24 .463 1 .00 11 .89 A N
ATOM 2945 C ASN A 396 2 .806 17 .197 24 .475 1 .00 13 .64 A C
ATOM 2946 O ASN A 396 2 .995 16 .743 25 .634 1 .00 14 .92 A O
ATOM 2947 N ASN A 397 2 .973 16 .452 23 .376 1 .00 13 .56 A N
ATOM 2948 CA ASN A 397 3 .539 15 .085 23 .451 1 .00 12 .37 A C
ATOM 2949 CB ASN A 397 2. .705 14 .080 22 .672 1 .00 12 .41 A C
ATOM 2950 CG ASN A 397 2 .539 14 .450 21 .192 1 .00 13 .07 A C
ATOM 2951 ODl ASN A 397 2 .243 15 .594 20 .849 1 .00 12 .07 A O
ATOM 2952 ND2 ASN A 397 2 .683 13 .466 20 .324 1 .00 13 .08 A N
ATOM 2953 C ASN A 397 5 .011 15 .077 23 .010 1 .00 13 .13 A C
ATOM 2954 O ASN A 397 5 .607 14 .010 22 .663 1 .00 11 .34 A O
ATOM 2955 N VAL A 398 5. .577 16 .291 23 .028 1 .00 12 .66 A N
ATOM 2956 CA VAL A 398 6 .992 16 .524 22 .914 1 .00 12 .58 A C
ATOM 2957 CB VAL A 398 7 .329 17 .261 21 .626 1 .00 13 .06 A C
ATOM 2958 CGI VAL A 398 8, .835 17 .523 21, .533 1 .00 11 .41 A C
ATOM 2959 CG2 VAL A 398 6 .846 16 .476 20 .408 1 .00 12 .98 A C
ATOM 2960 C VAL A 398 7 .381 17 .412 24 .105 1 .00 12 .92 A C
ATOM 2961 O VAL A 398 6, .819 18 .501 24 .272 1 .00 13 .08 A O
ATOM 2962 N GLU A 399 8, .288 16 .913 24 .945 1 .00 12 .47 A N
ATOM 2963 CA GLU A 399 8 .797 17 .666 26 .107 1 .00 12 .81 A C
ATOM 2964 CB GLU A 399 8 .339 17 .054 27 .452 1 .00 12 .40 A C
ATOM 2965 CG GLU A 399 6, .870 17 .340 27, .793 1 .00 11 .56 A C
ATOM 2966 CD GLU A 399 6, .538 17 .357 29 .284 1 .00 13 .58 A C
ATOM 2967 OEl GLU A 399 5 .312 17 .324 29 .635 1 .00 14 .48 A O
ATOM 2968 OE2 GLU A 399 7, .471 17 .421 30, .112 1, .00 14 .20 A O
ATOM 2969 C GLU A 399 10, .307 17 .680 26, .052 1 .00 12 .44 A C
ATOM 2970 O GLU A 399 10, .920 16 .624 25, .929 1 .00 13 .16 A O
ATOM 2971 N ASN A 400 10, .890 18 .883 26 .174 1. .00 12 .69 A N
ATOM 2972 CA ASN A 400 12, .326 19 .098 26, .073 1 .00 12 .36 A C
ATOM 2973 CB ASN A 400 12, .636 19 .953 24, .822 1 .00 12 .37 A C
ATOM 2974 CG ASN A 400 12, .185 19 .302 23 .559 1 .00 14 .26 A C
ATOM 2975 ODl ASN A 400 12, .621 18, .213 23, .247 1, .00 16 .94 A O
ATOM 2976 ND2 ASN A 400 11, .302 19 .964 22, ,817 1 .00 14 .56 A N
ATOM 2977 C ASN A 400 12, .959 19 .820 27, .254 1 .00 11 .55 A C
ATOM 2978 O ASN A 400 12, .363 20, .716 27. ,867 1, .00 11 .03 A O
ATOM 2979 N VAL A 401 14, ,200 19, .448 27, .543 1, .00 11 .83 A N
ATOM 2980 CA VAL A 401 15. .042 20 .183 28, .494 1 .00 11 .67 A C
ATOM- 2981 CB VAL A 401 15, .230 19, .394 29, ,804 1. .00 11. .24 A C
ATOM 2982 CGI VAL A 401 16. .317 20, .017 30. .668 1, ,00 13 .18 A C
ATOM 2983 CG2 VAL A 401 13, .962 19, .359 30, .558 1, .00 11 .36 A C
ATOM 2984 C VAL A 401 16. .351 20 .372 27, .792 1, .00 12 .10 A C
ATOM 2985 O VAL A 401 17. .022 19. ,394 27. ,471 1 , .00 12, .05 A O
ATOM 2986 N PHE A 402 16. .693 21, .634 27, .528 1, .00 12, .69 A N
ATOM 2987 CA PHE A 402 17. .841 22 , .019 26, ,744 1, ,00 14 .06 A C
ATOM 2988 CB PHE A 402 17. ,401 22 , .853 25, ,517 1. .00 14, .70 A C
ATOM 2989 CG PHE A 402 16. ,602 22, .079 24. ,464 1. .00 12, .42 A C
ATOM 2990 CDl PHE A 402 15. .936 22 , .764 23, ,455 1. .00 15, .18 A C
ATOM 2991 CEl PHE A 402 15. ,222 22. .069 22. ,468 1. ,00 12 , .37 A C
ATOM 2992 CZ PHE A 402 15. ,195 20, .700 22. ,489 1. .00 11, .68 A C
ATOM 2993 CE2 PHE A 402 15. .841 20, .022 23. ,493 1, .00 14, .06 A C
ATOM 2994 CD2 PHE A 402 16. .534 20, .698 24. ,465 1, .00 10, .95 A C
ATOM 2995 C PHE A 402 18. ,725 22. .896 27. ,641 1. ,00 15, .85 A C
ATOM 2996 O PHE A 402 18. ,356 24, .021 27. ,952 1. ,00 16, .80 A O
ATOM 2997 N ILE A 403 19. ,886 22 , ,379 28. ,028 1. .00 16, .60 A N
ATOM 2998 CA ILE A 403 20. ,787 23. ,062 28. 963 1. ,00 17, .41 A C
ATOM 2999 CB ILE A 403 21. ,088 22. .167 30. ,160 1. ,00 16, .74 A C
ATOM 3000 CGI ILE A 403 19. ,802 21, .886 30. ,944 1. .00 17, .16 A C
ATOM 3001 CDl ILE A 403 19. ,946 20, .733 31. .931 1. .00 15, .43 A C
ATOM 3002 CG2 ILE A 403 22. ,143 22 , .809 31. ,095 1. .00 17, ,20 A C ATOM 3003 C ILE A 403 22.064 23.395 28.240 1.00 17.62 A C
ATOM 3004 O ILE A 403 22 .812 22 .520 27 .854 1 .00 17 .97 A O
ATOM 3005 N ASN A 404 22 .299 24 .678 28 .026 1 .00 18 .39 A N
ATOM 3006 CA ASN A 404 23 .429 25 .112 27 .231 1 .00 19 .61 A C
ATOM 3007 CB ASN A 404 23 .255 26 .599 26 .874 1 .00 20 .94 A C
ATOM 3008 CG ASN A 404 24 .297 27 .071 25 .913 1 .00 26 .59 A C
ATOM 3009 ODl ASN A 404 24 .339 26 .618 24 .752 1 .00 32 .24 A O
ATOM 3010 ND2 ASN A 404 25 .177 27 .980 26 .381 1 .00 32 .91 A N
ATOM 3011 C ASN A 404 24 .773 24 .892 27 .940 1 .00 18 .94 A C
ATOM 3012 O ASN A 404 25 .769 24 .575 27 .296 1 .00 18 .69 A O
ATOM 3013 N ALA A 405 24 .779 25 .020 29 .262 1 .00 18 .60 A N
ATOM 3014 CA ALA A 405 26 .011 24 .902 30 .044 1 .00 19 .42 A C
ATOM 3015 CB ALA A 405 26 .450 26 .317 30 .582 1 .00 19 .40 A C
ATOM 3016 C ALA A 405 25 .787 23 .934 31 .217 1 .00 19 .12 A C
ATOM 3017 O ALA A 405 25 .582 24 .364 32 .360 1 .00 18 .74 A O
ATOM 3018 N PRO A 406 25 .782 22 .632 30 .936 1 .00 19 .16 A N
ATOM 3019 CA PRO A 406 25 .508 21 .629 31 .977 1. .00 19 .37 A C
ATOM 3020 CB PRO A 406 25 .266 20 .351 31 .156 1, .00 19 .25 A C
ATOM 3021 CG PRO A 406 26, .120 20 .546 29, .977 1 .00 19 .80 A C
ATOM 3022 CD PRO A 406 26, .033 22 .010 29, ,631 1, .00 18 .90 A C
ATOM 3023 C PRO A 406 26, .689 21 .437 32, .923 1, .00 19 .45 A C
ATOM 3024 O PRO A 406 27, .815 21 .833 32, .607 1, .00 19 .95 A O
ATOM 3025 N GLN A 407 26, .437 20 .819 34, .072 1. .00 19 .97 A N
ATOM 3026 CA GLN A 407 27, .490 20 .446 35, .016 1, .00 20 .20 A C
ATOM 3027 CB GLN A 407 26, .908 20. .387 36, .413 1, .00 21 .02 A C
ATOM 3028 CG GLN A 407 26 .155 21 .620 36, .805 1, .00 22 .32 A C
ATOM 3029 CD GLN A 407 25 .122 21 .323 37, .849 1 .00 23 .76 A C
ATOM 3030 OEl GLN A 407 25 .320 20 .443 38, .713 1, .00 21 .20 A 0
ATOM 3031 NE2 GLN A 407 24, .016 22 .040 37, .789 1. .00 22 .99 A N
ATOM 3032 C GLN A 407 28, .062 19 .075 34, .675 1, .00 20 .27 A C
ATOM 3033 O GLN A 407 27, .392 18 .232 34, .057 1, .00 19 .75 A O
ATOM 3034 N SER A 408 29, .294 18 .830 35, .099 1, .00 20 .30 A N
ATOM 3035 CA SER A 408 29, ,869 17 .491 35, .033 1, .00 20 .08 A C
ATOM 3036 CB SER A 408 31, .393 17 .538 35, ,212 1, .00 20 .55 A C
ATOM 3037 OG SER A 408 32, .042 18 .067 34, .072 1, .00 19 .34 A O
ATOM 3038 C SER A 408 29, .269 16 .615 36, .120 1. .00 19 .97 A C
ATOM 3039 O SER A 408 29, .130 17 .043 37. .268 1, .00 20 .96 A O
ATOM 3040 N GLY A 409 28, .980 15. .362 35. .775 1, .00 19 .80 A N
ATOM 3041 CA GLY A 409 28, .447 14 .392 36. .715 1, .00 18 .94 A C
ATOM 3042 C GLY A 409 27. .216 13, .697 36. .160 1. .00 19, .28 A C
ATOM 3043 O GLY A 409 27, .026 13, .646 34. ,940 1, .00 18 .32 A O
ATOM 3044 N THR A 410 26, .350 13, .224 37. .058 1, ,00 18 .72 A N
ATOM 3045 CA THR A 410 25. .226 12, .396 36. .678 1, .00 18, .26 A C
ATOM 3046 CB THR A 410 25. ,105 11, .220 37. .631 1, ,00 17, .98 A C
ATOM 3047 OGl THR A 410 26. .334 10, .466 37. .637 1. .00 16 .34 A O
ATOM 3048 CG2 THR A 410 24. .038 10, .227 37. ,136 1. .00 18, .99 A C
ATOM 3049 C THR A 410 23. .923 13. .183 36. ,687 1. .00 18, .19 A C
ATOM 3050 O THR A 410 23. .510 13, ,735 37. ,718 1. .00 18, .85 A 0
ATOM 3051 N TYR A 411 23. ,274 13, .241 35. ,524 1. .00 17, .34 A N
ATOM 3052 CA TYR A 411 21. .942 13 .783 35. .430 1, .00 16 .46 A C
ATOM 3053 CB TYR A 411 21, .731 14 .459 34. .067 1, .00 16 .84 A C
ATOM 3054 CG TYR A 411 22. .286 15 .869 34. .025 1. .00 16 .33 A C
ATOM 3055 CDl TYR A 411 21, .458 16, .953 34. ,156 1 , .00 16 .27 A C
ATOM 3056 CEl TYR A 411 21, ,956 18, .231 34. .131 1, .00 16 .41 A C
ATOM 3057 CZ TYR A 411 23. .319 18, .438 33, ,994 1. .00 17 .03 A C
ATOM 3058 OH TYR A 411 23. ,789 19, .744 34. ,031 1. ,00 17, .03 A O
ATOM 3059 CE2 TYR A 411 24. .172 17, .380 33. ,880 1. .00 16, .15 A C
ATOM 3060 CD2 TYR A 411 23. .660 16, .099 33. ,889 1. .00 17, .36 A C
ATOM 3061 C TYR A 411 20. .956 12, .627 35. ,606 1. .00 16, .14 A C
ATOM 3062 O TYR A 411 21. .157 11. .557 35. ,041 1. .00 15, .95 A 0
ATOM 3063 N THR A 412 19. .920 12, .841 36. ,399 1. .00 15, .50 A N
ATOM 3064 CA THR A 412 18. .760 11, .959 36. ,418 1. ,00 16, .39 A C
ATOM 3065 CB THR A 412 18. .107 12, .037 37. ,808 1. .00 16, .73 A C ATOM 3066 OGl THR A 412 19,.041 11,.544 38..783 1..00 16,.43 A O
ATOM 3067 CG2 THR A 412 16. .877 11, .115 37. ,946 1, .00 18. .16 A C
ATOM 3068 C THR A 412 17, ,764 12, .397 35. .344 1, .00 16. .26 A C
ATOM 3069 O THR A 412 17. .404 13, .568 35. .286 1, .00 15. .84 A O
ATOM 3070 N VAL A 413 17, .313 11, .444 34. .516 1, .00 16, .67 A N
ATOM 3071 CA VAL A 413 16, .342 11, .672 33, .452 1, .00 15, .75 A C
ATOM 3072 CB VAL A 413 16, .924 11, .246 32. .066 1, .00 16, .41 A C
ATOM 3073 CGI VAL A 413 15. .914 11, .476 30, .914 1, .00 15, .53 A C
ATOM 3074 CG2 VAL A 413 18. .240 11, .946 31, .773 1 .00 15, .42 A C
ATOM 3075 C VAL A 413 15, ,134 10, .811 33, .791 1 .00 16, .95 A C
ATOM 3076 O VAL A 413 15, .232 9, .574 33, .667 1 .00 18, .21 A O
ATOM 3077 N GLU A 414 14, .040 11, .439 34, .256 1 .00 16, .21 A N
ATOM 3078 CA GLU A 414 12, .803 10, .774 34. .736 1 .00 17, .01 A C
ATOM 3079 CB GLU A 414 12, .467 11, .202 36. .134 1, .00 16, .06 A C
ATOM 3080 CG GLU A 414 11. .518 10. .244 36. ,767 1. .00 18. .61 A C
ATOM 3081 CD GLU A 414 11. ,626 10, .318 38. ,265 1, ,00 17 , .99 A C
ATOM 3082 OEl GLU A 414 11. .212 11, .334 38. ,830 1, .00 20, .38 A O
ATOM 3083 OE2 GLU A 414 12, ,198 9, .404 38. ,810 1. .00 18, .55 A O
ATOM 3084 C GLU A 414 11, .715 11, ,142 33. ,729 1, .00 16, .05 A C
ATOM 3085 O GLU A 414 11. .428 12, .301 33. ,528 1, .00 15. .27 A O
ATOM 3086 N VAL A 415 11. .037 10, .172 33. ,145 1, .00 16. .93 A N
ATOM 3087 CA VAL A 415 9. .622 10, .184 32. ,847 1, .00 16, .73 A C
ATOM 3088 CB VAL A 415 9, .472 9, ,367 31, ,526 1, .00 17, .17 A C
ATOM 3089 CGI VAL A 415 8. .168 9, .660 30. .813 1, ,00 16, .00 A C
ATOM 3090 CG2 VAL A 415 10. .652 9, .660 30. .622 1, .00 16, .72 A C
ATOM 3091 C VAL A 415 8. .540 9, .769 33. ,787 1, .00 16. .71 A C
ATOM 3092 O VAL A 415 8, ,463 8, .634 34. .185 1, .00 16, .19 A O
ATOM 3093 N GLN A 416 7, .684 10, .736 34. .077 1, .00 16, .05 A N
ATOM 3094 CA GLN A 416 6, .553 10, .579 34. .989 1, .00 16, .43 A C
ATOM 3095 CB GLN A 416 6, .519 11. .747 35. .981 1, .00 16, .01 A C
ATOM 3096 CG GLN A 416 7. .786 11. .832 36. .802 1, .00 16, .27 A C
ATOM 3097 CD GLN A 416 7, .821 12 .929 37. .827 1, .00 17, .22 A C
ATOM 3098 OEl GLN A 416 6, .912 13. .762 37. .905 1, .00 16, .47 A O
ATOM 3099 NE2 GLN A 416 8, ,933 12, .972 38. .601 1, .00 16, .72 A N
ATOM 3100 C GLN A 416 5, .232 10, .504 34. .235 1, .00 17, .01 A C
ATOM 3101 O GLN A 416 4, .899 11, .388 33. .440 1, .00 16, .94 A O
ATOM 3102 N ALA A 417 4, .461 9, .462 34. .522 1, .00 17, .66 A N
ATOM 3103 CA ALA A 417 3. .122 9, .307 33, .953 1, .00 18, .17 A C
ATOM 3104 CB ALA A 417 2, ,770 7 .857 33. .891 1 .00 18, .16 A C
ATOM 3105 C ALA A 417 2, .092 10 .083 34. .790 1 .00 19 .19 A C
ATOM 3106 O ALA A 417 1. .542 9 .565 35. .775 1 .00 18 .61 A O
ATOM 3107 N TYR A 418 1 .859 11 .338 34. .437 1 .00 19 .27 A N
ATOM 3108 CA TYR A 418 0 .944 12 .153 35, .234 1 .00 20 .10 A C
ATOM 3109 CB TYR A 418 0 .985 13 .618 34, .803 1 .00 20 .65 A C
ATOM 3110 CG TYR A 418 0 .021 14 .496 35. ,570 1 .00 23 .36 A C
ATOM 3111 CDl TYR A 418 0 .255 14 .818 36, ,908 1 .00 26 .20 A C
ATOM 3112 CEl TYR A 418 -0 .645 15 .610 37, .625 1 .00 29 .96 A C
ATOM 3113 CZ TYR A 418 -1 .772 16 .099 36, ,990 1 .00 31 .45 A C
ATOM 3114 OH TYR A 418 -2 .659 16 .888 37, .685 1 .00 34 .45 A O
ATOM 3115 CE2 TYR A 418 -2 .018 15 .804 35. .652 1 .00 29 .32 A C
ATOM 3116 CD2 TYR A 418 -1 .123 15 .002 34, .957 1 .00 26 .35 A C
ATOM 3117 C TYR A 418 -0 .477 11 .623 35, .158 1 .00 19 .95 A c
ATOM 3118 O TYR A 418 -1 .142 11 .445 36, .190 1 .00 19 .83 A 0
ATOM 3119 N ASN A 419 -0 .928 11 .332 33, .945 1 .00 19 .39 A N
ATOM 3120 CA ASN A 419 -2 .284 10 .855 33, .708 1 .00 19 .40 A C
ATOM 3121 CB ASN A 419 -3 .243 12 .051 33, .629 1 .00 20 .03 A C
ATOM 3122 CG ASN A 419 -4 .705 11 .625 33 .611 1 .00 21 .53 A C
ATOM 3123 ODl ASN A 419 -5 .094 10 .758 34, ,354 1 .00 23 .42 A O
ATOM 3124 ND2 ASN A 419 -5 .493 12 .212 32 .727 1 .00 22 .67 A N
ATOM 3125 C ASN A 419 -2 .374 10 .079 32 .402 1 .00 19 .75 A C
ATOM 3126 O ASN A 419 -2 .186 10 .646 31 .317 1 .00 19 .24 A O
ATOM 3127 N VAL A 420 -2 .703 8 .795 32, ,486 1 .00 19 .24 A N
ATOM 3128 CA VAL A 420 -2 .744 7 .948 31, .295 1 .00 18 .57 A C ATOM 3129 CB VAL A 420 -1.533 6.986 31,.288 1.00 18.55 A C
ATOM 3130 CGI VAL A 420 -1 .504 6 .086 30, .040 1 .00 17 .15 A C
ATOM 3131 CG2 VAL A 420 -0 .196 7 .799 31, .402 1 .00 20 .43 A C
ATOM 3132 C VAL A 420 -4 .067 7 .165 31, .234 1 .00 18 .55 A C
ATOM 3133 O VAL A 420 -4 .109 5 .996 31, .606 1, .00 18 .55 A O
ATOM 3134 N PRO A 421 -5. .132 7 .816 30, .776 1, .00 18 .79 A N
ATOM 3135 CA PRO A 421 -6 .444 7 .169 30, .635 1, .00 19 .54 A C
ATOM 3136 CB PRO A 421 -7 .397 8 .324 30, .288 1, .00 19 .65 A C
ATOM 3137 CG PRO A 421 -6 .540 9 .394 29, .746 1, .00 19 .70 A C
ATOM 3138 CD PRO A 421 -5 .175 9 .239 30, .396 1, .00 18 .68 A C
ATOM 3139 C PRO A 421 -6 .507 6 .141 29, .530 1, .00 19 .68 A C
ATOM 3140 O PRO A 421 -7 .411 5 .318 29, .565 1, .00 20 .15 A o
ATOM 3141 N VAL A 422 -5 .592 6 .178 28, ,566 1. .00 19 .04 A N
ATOM 3142 CA VAL A 422 -5 .594 5 .180 27. .505 1, .00 18 .85 A C
ATOM 3143 CB VAL A 422 -5 .990 5 .781 26, .146 1 .00 18 .35 A C
ATOM 3144 CGI VAL A 422 -6 .200 4 .653 25, .091 1 .00 18 .91 A C
ATOM 3145 CG2 VAL A 422 -7 .264 6 .616 26, .285 1 .00 18 .28 A C
ATOM 3146 C VAL A 422 -4 .226 4 .509 27, .448 1 .00 19 .38 A C
ATOM 3147 O VAL A 422 -3 .435 4 .713 26, .505 1, .00 18 .38 A O
ATOM 3148 N GLY A 423 -3 .957 3 .707 28, .480 1, .00 19 .46 A N
ATOM 3149 CA GLY A 423 -2 .642 3 .150 28, .702 1, .00 19 .62 A C
ATOM 3150 C GLY A 423 -2 .510 1 .665 28, .496 1, .00 19 .72 A C
ATOM 3151 O GLY A 423 -3 .464 0 .954 28, .162 1, .00 21 .71 A O
ATOM 3152 N PRO A 424 -1 .307 1 .174 28. .695 1, .00 19 .10 A N
ATOM 3153 CA PRO A 424 -0. .142 1 .999 29. .040 1. .00 17 .55 A C
ATOM 3154 CB PRO A 424 0, .876 0 .969 29. .467 1. .00 18 .01 A C
ATOM 3155 CG PRO A 424 0 .510 -0 .258 28. .696 1, .00 19 .87 A C
ATOM 3156 CD PRO A 424 -0, .988 -0 .267 28. .649 1, .00 19 .26 A C
ATOM 3157 C PRO A 424 0, .396 2 .842 27. .899 1, .00 16 .87 A C
ATOM 3158 O PRO A 424 0, ,038 2, .660 26. .733 1, .00 17 .20 A O
ATOM 3159 N GLN A 425 1 .248 3 .798 28, .239 1, .00 15 .38 A N
ATOM 3160 CA GLN A 425 1 .848 4 .678 27, ,240 1, .00 14 .36 A C
ATOM 3161 CB GLN A 425 1 .507 6 .140 27, .559 1, .00 14 .88 A C
ATOM 3162 CG GLN A 425 2 .070 7 .202 26, .576 1, .00 14 .70 A C
ATOM 3163 CD GLN A 425 1 .512 7 .043 25. .180 1, .00 16 .71 A c
ATOM 3164 OEl GLN A 425 0 .321 7 .321 24. .956 1, .00 15 .27 A O
ATOM 3165 NE2 GLN A 425 2 .349 6 .580 24. ,235 1, ,00 11 .93 A N
ATOM 3166 C GLN A 425 3, .341 4 .470 27. .252 1, .00 13 .10 A C
ATOM 3167 O GLN A 425 3, .987 4 .662 28. ,283 1, .00 12 .18 A o
ATOM 3168 N THR A 426 3, .887 4 .036 26. ,112 1, .00 12 .88 A N
ATOM 3169 CA THR A 426 5. .320 4 .008 25. .913 1. .00 12 .90 A C
ATOM 3170 CB THR A 426 5, .737 2 .949 24. ,890 1. .00 12 .37 A C
ATOM 3171 OGl THR A 426 5, .134 3 .254 23. .626 1, .00 12 .70 A O
ATOM 3172 CG2 THR A 426 5, .232 1, .573 25. ,283 1. .00 13 .56 A C
ATOM 3173 C THR A 426 5, .796 5, .370 25, ,413 1. .00 13 .16 A C
ATOM 3174 O THR A 426 4 .986 6 .223 25. .037 1, .00 14 .38 A o
ATOM 3175 N PHE A 427 7, .115 5 .551 25, .401 1, .00 12 .86 A N
ATOM 3176 CA PHE A 427 7, .741 6 .823 25. ,036 1, .00 12 .44 A C
ATOM 3177 CB PHE A 427 7, .802 7 .778 26. .240 1, .00 12 .42 A C
ATOM 3178 CG PHE A 427 8. .612 7 .235 27. .366 1, .00 12 .82 A C
ATOM 3179 CDl PHE A 427 9, .988 7 .361 27. .365 1. .00 15 .21 A C
ATOM 3180 CEl PHE A 427 10, .768 6 .801 28. ,381 1 , ,00 15 .74 A C
ATOM 3181 CZ PHE A 427 10, .161 6 .102 29. .408 1 , .00 14 .88 A C
ATOM 3182 CE2 PHE A 427 8, .766 5 .987 29. ,427 1, .00 16 .39 A C
ATOM 3183 CD2 PHE A 427 8, ,000 6, .538 28. .407 1. .00 14 .33 A C
ATOM 3184 C PHE A 427 9. .149 6, .532 24. .549 1. ,00 12 .14 A C
ATOM 3185 O PHE A 427 9, .694 5, .444 24. .807 1. .00 11 .55 A o
ATOM 3186 N SER A 428 9, .721 7, .523 23. ,867 1. .00 11, .59 A N
ATOM 3187 CA SER A 428 11, .116 7, .528 23. ,480 1. .00 11 .99 A C
ATOM 3188 CB SER A 428 11, .292 7 , .463 21. .965 1. .00 12 .17 A C
ATOM 3189 OG SER A 428 10, .837 6, .219 21. .442 1. .00 12, .32 A o
ATOM 3190 C SER A 428 11 .804 8 .776 24, .031 1. .00 12 .57 A C
ATOM 3191 O SER A 428 11 .174 9 .829 24, ,263 1, .00 11 .91 A O ATOM 3192 N LEU A 429 13,.103 8.620 24,.278 1,.00 12.35 A N
ATOM 3193 CA LEU A 429 13, .950 9 .712 24, ,714 1. .00 12 .57 A C
ATOM 3194 CB LEU A 429 14, .508 9 .476 26, .135 1, .00 12 .84 A C
ATOM 3195 CG LEU A 429 13, .542 9 .648 27, .296 1, .00 13 .77 A C
ATOM 3196 CDl LEU A 429 14, .046 8 .907 28, ,520 1. .00 15 .69 A C
ATOM 3197 CD2 LEU A 429 13, .348 11 .110 27, ,609 1, ,00 15 .64 A C
ATOM 3198 C LEU A 429 15, .098 9 ,756 23, .768 1, .00 11 .82 A C
ATOM 3199 O LEU A 429 15, .593 8 .707 23, .372 1, .00 11 .44 A O
ATOM 3200 N ALA A 430 15 .532 10 .957 23, .405 1, .00 11 .55 A N
ATOM 3201 CA ALA A 430 16, .805 11 .139 22, .699 1, .00 11 .87 A C
ATOM 3202 CB ALA A 430 16 .581 11 .528 21, .235 1, ,00 12 .63 A C
ATOM 3203 C ALA A 430 17, .613 12 .215 23, .404 1, .00 12 .76 A C
ATOM 3204 O ALA A 430 17, .072 13 .256 23, .776 1, .00 12 .14 A O
ATOM 3205 N ILE A 431 18, .907 11 .943 23, .584 1, .00 12 .52 A N
ATOM 3206 CA ILE A 431 19, .813 12 .835 24, .287 1, .00 13 .32 A C
ATOM 3207 CB ILE A 431 20 .325 12 .179 25, .593 1, .00 13 .00 A C
ATOM 3208 CGI ILE A 431 19 .175 11 .882 26, .542 1, .00 14 .30 A C
ATOM 3209 CDl ILE A 431 19 .575 11 .061 27, .776 1, .00 16 .21 A C
ATOM 3210 CG2 ILE A 431 21 .292 13 .123 26, .288 1, .00 14 .86 A C
ATOM 3211 C ILE A 431 21 .005 13 .176 23, .392 1, .00 12 .92 A C
ATOM 3212 O ILE A 431 21 .728 12, .288 22, .937 1, ,00 11 .26 A O
ATOM 3213 N VAL A 432 21, .192 14, .464 23, .134 1, ,00 13 .80 A N
ATOM 3214 CA VAL A 432 22, .387 14, .966 22, .483 1, .00 15 .20 A C
ATOM 3215 CB VAL A 432 22, .028 15, ,996 21, .387 1, .00 15 .89 A C
ATOM 3216 CGI VAL A 432 23, .293 16, .591 20, .809 1, .00 15 .25 A C
ATOM 3217 CG2 VAL A 432 21, .167 15, .361 20, ,293 1, .00 15 .39 A C
ATOM 3218 C VAL A 432 23, .346 15, .634 23. .498 1, .00 16 .38 A C
ATOM 3219 O VAL A 432 22. .923 16, .472 24, .298 1, .00 16 .49 A O
ATOM 3220 N HIS A 433 24, .633 15. .257 23, .458 1, .00 17 .45 A N
ATOM 3221 CA HIS A 433 25, .669 15, .872 24, .306 1, .00 18, .55 A C
ATOM 3222 CB HIS A 433 25, .637 15, .240 25. .711 1, ,00 19 .21 A C
ATOM 3223 CG HIS A 433 26, .553 15, .885 26, ,707 1. .00 19 .32 A C
ATOM 3224 NDl HIS A 433 26, .497 17, .233 27, ,015 1, .00 18, .22 A N
ATOM 3225 CEl HIS A 433 27, .378 17, .497 27, .969 1, .00 18 .90 A C
ATOM 3226 NE2 HIS A 433 27, .999 16, .380 28, ,289 1. .00 16 .51 A N
ATOM 3227 CD2 HIS A 433 27, .502 15, .353 27, .513 1, .00 17 .65 A C
ATOM 3228 C HIS A 433 27, .031 15, .627 23. .684 1, ,00 19 .74 A C
ATOM 3229 O HIS A 433 27, .664 16, .546 23. .133 1, .00 21 .68 A O
ATOM 3230 OXT HIS A 433 27, .463 14, .480 23. .735 1, .00 19 .29 A O
TER 3230 HIS A 433
HETATM 3231 CA CA A 601 15, .429 35, .876 3, .369 1, .00 16 .92 A CA
HETATM 3232 CA CA A 602 3 .346 16, .597 30, ,346 1, .00 13 .45 A CA
HETATM 3233 CA CA A 603 9 .615 28, .353 34, ,891 1, .00 17 .30 A CA
ATOM 3234 N ASP B 16 3 .955 53 , .303 -10. .201 1, ,00 49 .01 B N
ATOM 3235 CA ASP B 16 4 .171 51, .870 -9. .771 1, .00 49 .32 B C
ATOM 3236 CB ASP B 16 5 .553 51, .425 -10. .270 1, .00 49 .78 B C
ATOM 3237 CG ASP B 16 6 .176 52, .438 -11. .248 1, .00 52 .12 B C
ATOM 3238 ODl ASP B 16 5 .667 52, .549 -12, ,399 1, .00 54 .86 B O
ATOM 3239 OD2 ASP B 16 7 .151 53, .181 -10, ,957 1, .00 52 .51 B O
ATOM 3240 C ASP B 16 4 .009 51, .690 -8, ,232 1, .00 48 .45 B C
ATOM 3241 O ASP B 16 4 .793 50, .996 -7, ,567 1, .00 47 .87 B O
ATOM 3242 N ARG B 17 2 .959 52, ,301 -7. .687 1, .00 47 .87 B N
ATOM 3243 CA ARG B 17 2 .863 52, .592 -6, .247 1, .00 47 .30 B C
ATOM 3244 CB ARG B 17 2 .430 54, .064 -6, .059 1. .00 46 .77 B C
ATOM 3245 CG ARG B 17 3 .107 55, .055 -7, .028 1, .00 44 .50 B C
ATOM 3246 CD ARG B 17 2 .860 56, .528 -6, .691 1, .00 39 .98 B C
ATOM 3247 NE ARG B 17 3 .266 56, .891 -5, .335 1, .00 33 .05 B N
ATOM 3248 CZ ARG B 17 4 .483 57, .334 -5, .001 1, .00 29 .36 B C
ATOM 3249 NHl ARG B 17 5 .440 57, .459 -5, .915 1, .00 28 .07 B N
ATOM 3250 NH2 ARG B 17 4 .752 57 .650 -3, .740 1, .00 24 .57 B N
ATOM 3251 C ARG B 17 1 .917 51 .699 -5, .415 1, .00 48 .01 B C
ATOM 3252 O ARG B 17 1 .463 52 .120 -4, .342 1 .00 47 .42 B O
ATOM 3253 N HIS B 18 1 .616 50 .486 -5, .885 1 .00 48 .51 B N ATOM 3254 CA HIS B 18 0.770 49.573 -5.108 1.00 48.98 B C
ATOM 3255 CB HIS B 18 0 .515 48 .266 -5 .875 1 .00 49 .29 B C
ATOM 3256 CG HIS B 18 -0 .510 48 .388 -6 .961 1 .00 50 .05 B C
ATOM 3257 NDl HIS B 18 -0. .195 48 .803 -8 .238 1 .00 51 .24 B N
ATOM 3258 CEl HIS B 18 -1 .291 48 .814 -8 .979 1 .00 51 .27 B C
ATOM 3259 NE2 HIS B 18 -2 .305 48 .419 -8, .228 1 .00 50 .69 B N
ATOM 3260 CD2 HIS B 18 -1. .844 48 .147 -6, .962 1 .00 50 .64 B C
ATOM 3261 C HIS B 18 1. .429 49 .229 -3, .770 1 .00 49 .16 B C
ATOM 3262 O HIS B 18 2 .598 48 .822 -3, .738 1 .00 49 .03 B O
ATOM 3263 N ASN B 19 0 .690 49 .386 -2, .667 1 .00 49 .15 B N
ATOM 3264 CA ASN B 19 1 .167 48 .868 -1, .384 1 .00 49 .11 B C
ATOM 3265 CB ASN B 19 0 .276 49 .313 -0, .205 1 .00 49 .60 B C
ATOM 3266 CG ASN B 19 0, .951 49 .099 1, .176 1 .00 51 .24 B C
ATOM 3267 ODl ASN B 19 0 .415 48 .415 2, .058 1 .00 53 .59 B O
ATOM 3268 ND2 ASN B 19 2 .123 49 .705 1 .363 1 .00 54 .56 B N
ATOM 3269 C ASN B 19 1 .241 47 .332 -1 .459 1 .00 48 .22 B C
ATOM 3270 O ASN B 19 0 .443 46 .685 -2 .138 1 .00 47 .43 B O
ATOM 3271 N LEU B 20 2 .221 46 .772 -0 .762 1 .00 47 .20 B N
ATOM 3272 CA LEU B 20 2 .393 45 .333 -0 .689 1 .00 46 .38 B C
ATOM 3273 CB LEU B 20 3 .743 45 .000 -0 .055 1 .00 46 .97 B C
ATOM 3274 CG LEU B 20 4 .896 45 .800 -0 .684 1 .00 48 .40 B C
ATOM 3275 CDl LEU B 20 6. .201 45 .666 0 .117 1 .00 49 .54 B C
ATOM 3276 CD2 LEU B 20 5 .076 45 .391 -2, .158 1 .00 48 .69 B C
ATOM 3277 C LEU B 20 1 .235 44 .792 0, .141 1 .00 44 .75 B C
ATOM 3278 O LEU B 20 1 .113 45 .092 1, .342 1 .00 45 .26 B O
ATOM 3279 N LYS B 21 0 .338 44 .073 -0, .523 1 .00 41 .97 B N
ATOM 3280 CA LYS B 21 -0 .740 43 .395 0. .170 1 .00 39 .98 B C
ATOM 3281 CB LYS B 21 -2 .088 44 .025 -0, .183 1 .00 40 .54 B C
ATOM 3282 CG LYS B 21 -3 .225 43 .550 0, .700 1 .00 41 .63 B C
ATOM 3283 CD LYS B 21 -4 .257 44 .620 0, .878 1 .00 43 .44 B C
ATOM 3284 CE LYS B 21 -5, .391 44 .131 1, .718 1 .00 44 .82 B C
ATOM 3285 NZ LYS B 21 -4, .992 44, .004 3, .147 1 .00 47 .17 B N
ATOM 3286 C LYS B 21 -0, .710 41 .917 -0, .214 1 .00 37 .09 B C
ATOM 3287 O LYS B 21 -0, .679 41, .588 -1, ,395 1 .00 35 .80 B O
ATOM 3288 N THR B 22 -0, .685 41, .045 0. .796 1 .00 33 .91 B N
ATOM 3289 CA THR B 22 -0. .642 39, .592 0. .593 1. .00 31, .44 B C
ATOM 3290 CB THR B 22 0, .734 39, .046 1, .030 1. .00 31, .70 B C
ATOM 3291 OGl THR B 22 1, .002 39. .436 2. .387 1. .00 31 .47 B O
ATOM 3292 CG2 THR B 22 1, ,857 39. .681 0. .211 1, .00 31, .30 B C
ATOM 3293 C THR B 22 -1, .739 38, ,843 1. .342 1, .00 29, .57 B C
ATOM 3294 O THR B 22 -1, .830 37, .617 1. .246 1, .00 28, .17 B O
ATOM 3295 N GLU B 23 -2, .542 39, .576 2. .107 1, .00 27, .46 B N
ATOM 3296 CA GLU B 23 -3, .672 39, ,011 2. .828 1, .00 27, .22 B C
ATOM 3297 CB : BGLU B 23 -3. .280 38. .728 4. .282 0, .50 27, .51 B C
ATOM 3298 CB . AGLU B 23 -3, .287 38. .646 4. .277 0, ,50 27, .17 B C
ATOM 3299 CG : BGLU B 23 -2. .826 37, ,304 4. .512 0, .50 29, .10 B C
ATOM 3300 CG . AGLU B 23 -3, .050 39. .822 5. .223 0, .50 27, .55 B C
ATOM 3301 CD : BGLU B 23 -2, .236 37, .062 5. .891 0, ,50 30, .84 B C
ATOM 3302 CD ; AGLU B 23 -3, ,020 39, .396 6. .689 0. .50 28, ,07 B C
ATOM 3303 OE1BGLU B 23 -1, .959 38, .040 6, ,614 0. .50 32, .02 B O
ATOM 3304 OE1AGLU B 23 -2. .853 38, .186 6, ,954 0, .50 28, .41 B O
ATOM 3305 OE2BGLU B 23 -2. ,054 35, .879 6. ,241 0 , .50 31, .16 B O
ATOM 3306 OE2AGLU B 23 -3. .182 40, .264 7. ,579 0. .50 28, .60 B O
ATOM 3307 C GLU B 23 -4. .842 39, .988 2, ,799 1. .00 25. .94 B C
ATOM 3308 O GLU B 23 -4. .631 41. .199 2. .805 1 , ,00 25. .17 B 0
ATOM 3309 N TRP B 24 -6. .065 39. ,462 2. ,765 1. .00 24. .57 B N
ATOM 3310 CA TRP B 24 -7. ,264 40. .300 2. ,708 1. .00 23. .89 B C
ATOM 3311 CB TRP B 24 -7. ,910 40. .174 1. ,304 1, .00 23. .59 B C
ATOM 3312 CG TRP B 24 -7. ,105 40. .786 0. ,245 1. ,00 21, .71 B C
ATOM 3313 CDl TRP B 24 -7. ,232 42. .050 -0. ,239 1. .00 21, .02 B C
ATOM 3314 NEl TRP B 24 -6. ,293 42. ,276 -1. ,211 1. .00 18. .62 B N
ATOM 3315 CE2 TRP B 24 -5, .544 41, .148 -1. .396 1, ,00 19 .02 B C
ATOM 3316 CD2 TRP B 24 -6, .006 40, .190 -0. .480 1, .00 20 .72 B C ATOM 3317 CE3 TRP B 24 -5.387 38.941 -0.454 1.00 19.49 β C
ATOM 3318 CZ3 TRP B 24 -4 .326 38 .694 -1 .313 1 .00 20 .25 B C
ATOM 3319 CH2 TRP B 24 -3 .883 39 .662 -2 .207 1 .00 21 .63 B C
ATOM 3320 CZ2 TRP B 24 -4 .477 40 .911 -2 .257 1 .00 22 .09 B C
ATOM 3321 C TRP B 24 -8 .294 39 .948 3 .789 1 .00 23 .91 B C
ATOM 3322 O TRP B 24 -9 .369 39 .456 3 .467 1 .00 22 .74 B O
ATOM 3323 N PRO B 25 -7 .986 40 .196 5 .070 1 .00 24 .64 B N
ATOM 3324 CA PRO B 25 -8 .918 39 .850 6 .161 1 .00 25 .12 B C
ATOM 3325 CB PRO B 25 -8 .176 40 .312 7 .448 1 .00 25 .87 B C
ATOM 3326 CG PRO B 25 -7 .011 41 .163 7 .002 1 .00 26 .08 B C
ATOM 3327 CD PRO B 25 -6 .737 40 .807 5 .562 1 .00 25 .49 B C
ATOM 3328 C PRO B 25 -10 .307 40 .520 6. .029 1 .00 25 .18 B C
ATOM 3329 O PRO B 25 -11 .310 39 .978 6 .469 1 .00 24 .94 B O
ATOM 3330 N GLU B 26 -10 .350 41 .668 5 .364 1 .00 25 .43 B N
ATOM 3331 CA GLU B 26 -11 .581 42 .416 5 .141 1 .00 25 .93 B C
ATOM 3332 CB GLU B 26 -11 .243 43 .829 4 .627 1 .00 26 .70 B C
ATOM 3333 CG GLU B 26 -10 .690 43 .922 3 .189 1 .00 28 .62 B C
ATOM 3334 CD GLU B 26 -9 .169 43 .775 3 .077 1 .00 29 .61 B C
ATOM 3335 OEl GLU B 26 -8 .535 43 .174 3 .985 1 .00 28 .68 B O
ATOM 3336 OE2 GLU B 26 -8 .608 44 .252 2 .057 1 .00 31 .71 B O
ATOM 3337 C GLU B 26 -12 .571 41 .705 4 .193 1 .00 25 .60 B C
ATOM 3338 O GLU B 26 -13 .746 42 .060 4 .139 1 .00 24 .75 B O
ATOM 3339 N LEU B 27 -12 .119 40 .672 3 .483 1. .00 24 .52 B N
ATOM 3340 CA LEU B 27 -12 .957 40 .024 2 .483 1 .00 23 .54 B C
ATOM 3341 CB LEU B 27 -12, .104 39 .593 1, .287 1 .00 23 .84 B C
ATOM 3342 CG LEU B 27 -11, .506 40 .722 0 .430 1 .00 23 .08 B C
ATOM 3343 CDl LEU B 27 -10 .702 40 .165 -0 .732 1 .00 22 .31 B C
ATOM 3344 CD2 LEU B 27 -12, .603 41 .624 -0 .097 1 .00 23 .37 B C
ATOM 3345 C LEU B 27 -13, .716 38 .829 3, .042 1 .00 23 .75 B C
ATOM 3346 O LEU B 27 -14. .504 38 .205 2, .334 1 .00 23 .33 B O
ATOM 3347 N VAL B 28 -13. .490 38 .504 4, .312 1 .00 23 .89 B N
ATOM 3348 CA VAL B 28 -14, ,143 37 .357 4, .918 1 .00 24 .64 B C
ATOM 3349 CB VAL B 28 -13, .571 37 .050 6. .359 1 .00 24 .78 B C
ATOM 3350 CGI VAL B 28 -14, .359 35 .963 7, .027 1 .00 25. .39 B C
ATOM 3351 CG2 VAL B 28 -12, .099 36 .634 6, .272 1 .00 25. .78 B C
ATOM 3352 C VAL B 28 -15, .612 37 .694 4. .992 1 .00 24, .75 B C
ATOM 3353 O VAL B 28 -15. .952 38 .791 5, ,424 1, .00 25, .68 B O
ATOM 3354 N GLY B 29 -16. .468 36 .797 4, ,516 1, .00 24. .86 B N
ATOM 3355 CA GLY B 29 -17. .916 37 .000 4 , .539 1, .00 24. .80 B C
ATOM 3356 C GLY B 29 -18, .493 37 .638 3, .274 1, .00 25, .07 B C
ATOM 3357 O GLY B 29 -19, .692 37 .598 3, , 061 1, .00 25, .27 B O
ATOM 3358 N LYS B 30 -17, ,630 38 .203 2, .429 1, .00 25, .21 B N
ATOM 3359 CA LYS B 30 -18, .025 38 .782 1, .146 1, .00 24 , .62 B C
ATOM 3360 CB LYS B 30 -16, .952 39 .780 0. ,679 1, .00 25, .65 B C
ATOM 3361 CG LYS B 30 -16, ,716 40, .964 1. ,606 1, .00 28, .42 B C
ATOM 3362 CD LYS B 30 -16. .577 42 .245 0. .785 1, .00 34, ,26 B C
ATOM 3363 CE LYS B 30 -16. ,462 43, .527 1. .631 1, .00 35, .92 B C
ATOM 3364 NZ LYS B 30 -15. ,996 43, .273 3. ,011 1, .00 37, .72 B N
ATOM 3365 C LYS B 30 -18. .188 37, .728 0. .065 1, .00 23 , ,30 B C
ATOM 3366 O LYS B 30 -17. .670 36, .623 0. .166 1, .00 22 , .29 B O
ATOM 3367 N SER B 31 -18. .884 38, .089 -1. .001 1, ,00 21. .94 B N
ATOM 3368 CA SER B 31 -19. ,036 37, ,204 -2. .145 1. .00 20. .89 B C
ATOM 3369 CB SER B 31 -20. ,046 37, .776 -3. .143 1. .00 21, .21 B C
ATOM 3370 OG SER B 31 -19. ,519 38, .912 -3. ,815 1. ,00 20. .40 B O
ATOM 3371 C SER B 31 -17. ,726 37, .017 -2. ,865 1. ,00 19. ,88 B C
ATOM 3372 O SER B 31 -16. ,828 37, .843 -2. ,800 1. .00 18. .67 B O
ATOM 3373 N VAL B 32 -17. ,649 35, .920 -3. ,588 1. .00 20. .34 B N
ATOM 3374 CA VAL B 32 -16. ,487 35. .617 -4. ,393 1. .00 20. .89 B C
ATOM 3375 CB ] BVAL B 32 -16. ,717 34 , .256 -5. ,141 0. ,50 20. .94 B C
ATOM 3376 CB AVAL B 32 -16. ,555 34. ,234 -5. ,043 0. ,50 21. ,07 B C
ATOM 3377 CGIBVAL B 32 -16. ,023 34. .221 -6. ,524 0. .50 20. .85 B C
ATOM 3378 CGIAVAL B 32 -17, .648 34 .180 -6, .069 0, .50 20, .79 B C
ATOM 3379 CG2BVAL B 32 -16, ,276 33 .087 -4. .281 0, .50 20, .73 B C ATOM 3380 CG2AVAL B 32 -15.193 33.903 -5..657 0.50 21.36 B C
ATOM 3381 C VAL B 32 -16 .238 36 .732 -5. .431 1. .00 20 .83 B C
ATOM 3382 O VAL B 32 -15 .100 37 .105 -5, .681 1 .00 20 .37 B O
ATOM 3383 N GLU B 33 -17 .316 37 .263 -6, .011 1. .00 21 .19 B N
ATOM 3384 CA GLU B 33 -17 .205 38 .264 -7, .072 1 .00 21 .20 B C
ATOM 3385 CB GLU B 33 -18 .553 38 .478 -7, .767 1 .00 21 .59 B C
ATOM 3386 CG GLU B 33 -19 .045 37 .271 -8, .543 1 .00 24 .67 B C
ATOM 3387 CD GLU B 33 -19 .799 36 .219 -7, .708 1 .00 29 .01 B C
ATOM 3388 OEl GLU B 33 -20 .001 35 .123 -8, .275 1 .00 36 .54 B O
ATOM 3389 OE2 GLU B 33 -20 .187 36 .437 -6, .517 1 .00 27 .78 B O
ATOM 3390 C GLU B 33 -16 .688 39 .571 -6, .497 1 .00 20 .62 B C
ATOM 3391 O GLU B 33 -15 .885 40 .255 -7, .130 1 .00 20 .20 B O
ATOM 3392 N GLU B 34 -17 .124 39 .910 -5, .283 1 .00 20 .61 B N
ATOM 3393 CA GLU B 34 -16 .627 41 .131 -4, .634 1 .00 20 .97 B C
ATOM 3394 CB GLU B 34 -17. .456 41 .533 -3 , .407 1. .00 21, .10 B C
ATOM 3395 CG GLU B 34 -18. .778 42 .224 -3. .722 1, .00 25, .82 B C
ATOM 3396 CD GLU B 34 -18. .615 43 .546 -4. .481 1, .00 31, .16 B C
ATOM 3397 OEl GLU B 34 -17. .968 44 .484 -3, .932 1, .00 32, .84 B 0
ATOM 3398 OE2 GLU B 34 -19, .135 43 .645 -5, .626 1. .00 33. .71 B O
ATOM 3399 C GLU B 34 -15, .156 40 .951 -4. .257 1. .00 19, .67 B C
ATOM 3400 O GLU B 34 -14, .340 41 .858 -4. .438 1, .00 18, .91 B O
ATOM 3401 N ALA B 35 -14, .809 39 .775 -3. ,765 1. .00 19. .75 B N
ATOM 3402 CA ALA B 35 -13, .414 39 .485 -3. .401 1, .00 19. .10 B C
ATOM 3403 CB ALA B 35 -13, .311 38 .127 -2. .749 1. .00 19, .66 B C
ATOM 3404 C ALA B 35 -12, .457 39 .581 -4. .582 1. .00 18, .90 B c
ATOM 3405 O ALA B 35 -11, ,387 40 .183 -4. .470 1, .00 18, .82 B O
ATOM 3406 N LYS B 36 -12, .839 38 .993 -5, ,716 1, .00 18 , .68 B N
ATOM 3407 CA LYS B 36 -11, .991 38 .978 -6, .894 1, .00 18, .17 B c
ATOM 3408 CB LYS B 36 -12, ,659 38 .220 -8, .063 1, .00 18, .44 B c
ATOM 3409 CG LYS B 36 -12. ,714 36 .693 -7. ,928 1. .00 19, .56 B c
ATOM 3410 CD LYS B 36 -13. .304 36 .026 -9. ,159 1, .00 20, .72 B c
ATOM 3411 CE LYS B 36 -13, .194 34 .496 -9. .136 1, ,00 22, .32 B c
ATOM 3412 NZ LYS B 36 -13, .963 33 .865 -10. .274 1, .00 20, .54 B N
ATOM 3413 C LYS B 36 -11, .648 40 .406 -7. ,316 1. .00 17 , .67 B c
ATOM 3414 O LYS B 36 -10, .500 40 .694 -7. .681 1, .00 17, .81 B O
ATOM 3415 N LYS B 37 -12, .614 41 .316 -7, .254 1, .00 17, .52 B N
ATOM 3416 CA LYS B 37 -12, .345 42 .667 -7, .746 1. .00 17, .89 B c
ATOM 3417 CB LYS B 37 -13, .621 43 .519 -7, .870 1, .00 17, .35 B c
ATOM 3418 CG LYS B 37 -14, .544 43 .165 -9, .036 1, .00 17, .43 B c
ATOM 3419 CD LYS B 37 -15, .847 44 .074 -9, .064 1, .00 15, .06 B c
ATOM 3420 CE LYS B 37 -16, .801 43 .812 -7, .921 1, .00 16 .48 B c
ATOM 3421 NZ LYS B 37 -18, .031 44 .685 -7, .989 1 .00 15 .66 B N
ATOM 3422 C LYS B 37 -11, .333 43 .372 -6, .852 1, .00 18 .04 B c
ATOM 3423 O LYS B 37 -10, .499 44 .126 -7, ,354 1 .00 18 .33 B O
ATOM 3424 N VAL B 38 -11, ,436 43 .174 -5, .535 1 .00 17 .93 B N
ATOM 3425 CA VAL B 38 -10, .525 43 .824 -4. .595 1 .00 18 .53 B C
ATOM 3426 CB VAL B 38 -11, .024 43 .636 -3 , ,136 1 .00 19 .32 B C
ATOM 3427 CGI VAL B 38 -9, .975 44 .055 -2, .128 1 .00 21 .52 B c
ATOM 3428 CG2 VAL B 38 -12, .310 44 .445 -2, .919 1 .00 20 .73 B c
ATOM 3429 C VAL B 38 -9, .122 43 .270 -4, ,742 1 .00 18 .91 B c
ATOM 3430 O VAL B 38 -8, .135 44 .013 -4, ,797 1 .00 17 .79 B 0
ATOM 3431 N ILE B 39 -9, .033 41 .947 -4, .830 1 .00 19 .11 B N
ATOM 3432 CA ILE B 39 -7, .747 41 .304 -5, .009 1 .00 19 .64 B C
ATOM 3433 CB ILE B 39 -7, .919 39 .764 -4, .973 1 .00 19 .43 B C
ATOM 3434 CGI ILE B 39 -8, .288 39 .324 -3, .573 1 .00 20 .37 B C
ATOM 3435 CDl ILE B 39 -8, .994 37 .995 -3, ,564 1 .00 21 .36 B C
ATOM 3436 CG2 ILE B 39 -6. .657 39 .024 -5, ,470 1 .00 19 .76 B C
ATOM 3437 C ILE B 39 -7, ,077 41 .759 -6, .287 1 .00 19 .71 B C
ATOM 3438 O ILE B 39 -5. .877 42 .087 -6, .266 1 .00 19 .70 B 0
ATOM 3439 N LEU B 40 -7, .816 41 .785 -7 , .404 1 .00 19 .22 B N
ATOM 3440 CA LEU B 40 -7, .205 42 .231 -8, .664 1 .00 19 .62 B C
ATOM 3441 CB LEU B 40 -8. .100 41 .927 -9. .888 1, .00 19, .18 B C
ATOM 3442 CG LEU B 40 -8. .145 40 .416 -10. .190 1 .00 20, .23 B C ATOM 3443 CDl LEU B 40 -9.235 40.047 -11.123 1.00 19.43 B C
ATOM 3444 CD2 LEU B 40 -6 .799 39 .947 -10 .725 1 .00 21 .26 B C
ATOM 3445 C LEU B 40 -6 .840 43 .716 -8 .608 1 .00 19 .20 B C
ATOM 3446 O LEU B 40 -5 .939 44 .144 -9 .300 1 .00 19 .48 B O
ATOM 3447 N GLN B 41 -7 .553 44 .494 -7 .803 1 .00 19 .70 B N
ATOM 3448 CA GLN B 41 -7 .216 45 .914 -7 .622 1 .00 20 .69 B C
ATOM 3449 CB GLN B 41 -8 .286 46 .641 -6 .813 1 .00 20 .30 B C
ATOM 3450 CG GLN B 41 -8 .068 48 .173 -6 .731 1 .00 20 .90 B C
ATOM 3451 CD GLN B 41 -8 .159 48 .842 -8 .083 1 .00 21 .54 B C
ATOM 3452 OEl GLN B 41 -9 .070 48 .529 -8 .858 1 .00 23 .88 B O
ATOM 3453 NE2 GLN B 41 -7 .224 49 .762 -8 .384 1 .00 20 .95 B N
ATOM 3454 C GLN B 41 -5 .880 46 .050 -6 .906 1 .00 22 .10 B C
ATOM 3455 O GLN B 41 -5 .105 46 .941 -7 .213 1 .00 22 .64 B O
ATOM 3456 N ASP B 42 -5 .625 45 .149 -5 .955 1 .00 22 .70 B N
ATOM 3457 CA ASP B 42 -4 .396 45 .178 -5 .161 1 .00 23 .28 B C
ATOM 3458 CB ASP B 42 -4 .654 44 .531 -3 .800 1 .00 22 .77 B C
ATOM 3459 CG ASP B 42 -5 .531 45 .369 -2 .928 1. .00 24 .12 B C
ATOM 3460 ODl ASP B 42 -5 .619 46 .599 -3 .174 1. .00 27 .20 B O
ATOM 3461 OD2 ASP B 42 -6 .206 44 .899 -1 .991 1, .00 25 .78 B O
ATOM 3462 C ASP B 42 -3 .273 44 .438 -5 .859 1. .00 23 .19 B C
ATOM 3463 O ASP B 42 -2 .103 44 .761 -5 .700 1, .00 24 .27 B O
ATOM 3464 N LYS B 43 -3 .629 43 .444 -6 .655 1, .00 22 .96 B N
ATOM 3465 CA LYS B 43 -2 .634 42 .541 -7 .203 1. .00 23 .10 B C
ATOM 3466 CB LYS B 43 -2 .508 41 .291 -6, .299 1, .00 23 .07 B C
ATOM 3467 CG LYS B 43 -1 .376 40 .306 -6, .701 1, .00 23 .16 B C
ATOM 3468 CD LYS B 43 -1 .348 39 .100 -5, .750 1. .00 24 .15 B C
ATOM 3469 CE LYS B 43 -0 .391 37 .996 -6 .217 1 .00 25 .40 B C
ATOM 3470 NZ LYS B 43 1 .031 38 .403 -6 .170 1. .00 25 .72 B N
ATOM 3471 C LYS B 43 -3 .067 42 .157 -8 .593 1 .00 23 .00 B C
ATOM 3472 O LYS B 43 -3 .672 41 .107 -8, .782 1 .00 22 .17 B O
ATOM 3473 N PRO B 44 -2 .772 43 .010 -9, .571 1 .00 24 .07 B N
ATOM 3474 CA PRO B 44 -3, .282 42 .826 -10, .948 1. .00 24 .35 B C
ATOM 3475 CB PRO B 44 -2 .632 43 .985 -11. .735 1, .00 24 .65 B C
ATOM 3476 CG PRO B 44 -2, .197 44 .997 -10. .702 1, .00 25 .50 B C
ATOM 3477 CD PRO B 44 -1, .960 44, .238 -9. .415 1, .00 24 .73 B C
ATOM 3478 C PRO B 44 -2, .929 41, .486 -11. .583 1, .00 24 .65 B C
ATOM 3479 O PRO B 44 -3, .680 40 , ,967 -12. .409 1, .00 25. .49 B O
ATOM 3480 N GLU B 45 -1. ,778 40. .935 -11. .206 1, .00 25, ,20 B N
ATOM 3481 CA GLU B 45 -1. ,310 39. .651 -11. ,725 1. .00 25. .70 B C
ATOM 3482 CB GLU B 45 0, ,226 39, ,599 -11. ,602 1. .00 26. .83 B C
ATOM 3483 CG GLU B 45 0. .764 39. .243 -10. ,206 1, .00 28, ,74 B C
ATOM 3484 CD GLU B 45 0. .925 40, .423 -9. ,262 1. .00 32 , .92 B C
ATOM 3485 OEl GLU B 45 1. .667 40, .252 -8. ,253 1. .00 34, .10 B O
ATOM 3486 OE2 GLU B 45 0. .316 41, .511 -9. ,488 1. ,00 33 , .16 B O
ATOM 3487 C GLU B 45 -1. .945 38. .404 -11. ,048 1. ,00 25 , ,32 B C
ATOM 3488 O GLU B 45 -1. .679 37. .270 -11. ,452 1. ,00 25. .52 B O
ATOM 3489 N ALA B 46 -2. .788 38 , .593 -10. .034 1. ,00 24 , .59 B N
ATOM 3490 CA ALA B 46 -3. ,327 37. .441 -9. ,309 1. ,00 23, .90 B C
ATOM 3491 CB ALA B 46 -4. ,271 37. .895 -8. ,229 1. ,00 23. .95 B C
ATOM 3492 C ALA B 46 -4, .015 36, .426 -10. .216 1, .00 23, .75 B C
ATOM 3493 O ALA B 46 -4, .777 36. .788 -11. .103 1, .00 22, .67 B O
ATOM 3494 N GLN B 47 -3, .717 35, .150 -9. ,982 1, .00 23, ,77 B N
ATOM 3495 CA GLN B 47 -4, ,438 34, .035 -10. ,568 1. .00 24. .59 B C
ATOM 3496 CB GLN B 47 -3, .479 32 , .976 -11. ,105 1. .00 25, .29 B C
ATOM 3497 CG GLN B 47 -2. ,425 33 , .498 -12. .080 1. .00 28, .88 B C
ATOM 3498 CD GLN B 47 -3. .025 33 , ,975 -13, ,393 1. .00 35, .56 B C
ATOM 3499 OEl GLN B 47 -3. .624 33 , ,176 -14, , 144 1. .00 40. .01 B O
ATOM 3500 NE2 GLN B 47 -2. .869 35. .278 -13. ,686 1. .00 38. .46 B N
ATOM 3501 C GLN B 47 -5. .298 33. .425 -9. ,460 1. .00 23. .84 B C
ATOM 3502 O GLN B 47 -4. .786 32. .790 -8. ,517 1. .00 24. .12 B O
ATOM 3503 N ILE B 48 -6. .597 33. .644 -9. ,559 1. .00 22. .91 B N
ATOM 3504 CA ILE B 48 -7. .502 33. .329 -8. ,463 1. .00 23. .01 B C
ATOM 3505 CB ILE B 48 -8. ,486 34. .462 -8. .235 1. .00 22, .88 B C ATOM 3506 CGI ILE B 48 -7.708 35.747 -7.988 1.00 22.10 B C
ATOM 3507 CDl ILE B 48 -8 .568 36 .992 -7 .917 1 .00 22 .38 B C
ATOM 3508 CG2 ILE B 48 -9 .391 34 .161 -7 ,036 1 .00 21 .57 B C
ATOM 3509 C ILE B 48 -8 .230 32 .047 -8 .746 1 .00 23 .69 B C
ATOM 3510 O ILE B 48 -8 .685 31 .820 -9 .877 1 .00 23 .31 B O
ATOM 3511 N ILE B 49 -8 .277 31 .206 -7. .716 1 .00 23 .55 B N
ATOM 3512 CA ILE B 49 -8 .894 29 .894 -7 .746 1 .00 24 .95 B C
ATOM 3513 CB ILE B 49 -7 .803 28 .812 -7, .480 1 .00 26 .22 B C
ATOM 3514 CGI ILE B 49 -6 .723 28 .868 -8, .575 1 .00 29 .02 B C
ATOM 3515 CDl ILE B 49 -7 .264 28 .733 -9, .982 1 .00 29 .18 B C
ATOM 3516 CG2 ILE B 49 -8 .409 27 .422 -7, .364 1 .00 28 .74 B C
ATOM 3517 C ILE B 49 -9 .903 29 .851 -6, .610 1 .00 23 .74 B C
ATOM 3518 O ILE B 49 -9 .620 30 .348 -5, .511 1 .00 24 .09 B O
ATOM 3519 N VAL B 50 -11 .045 29 .224 -6, .847 1 .00 22 .75 B N
ATOM 3520 CA VAL B 50 -12 .088 29 .110 -5 .838 1 .00 22 .00 B C
ATOM 3521 CB VAL B 50 -13 .441 29 .682 -6 .364 1 .00 21 .55 B C
ATOM 3522 CGI VAL B 50 -14 .583 29 .378 -5 .388 1 .00 22 .01 B C
ATOM 3523 CG2 VAL B 50 -13 .338 31 .190 -6 .581 1 .00 21 .04 B C
ATOM 3524 C VAL B 50 -12 .273 27 .639 -5, .439 1 .00 21 .99 B C
ATOM 3525 O VAL B 50 -12 .375 26 .780 -6, .291 1 .00 21 .98 B O
ATOM 3526 N LEU B 51 -12 .318 27 .363 -4, .141 1 .00 21 .70 B N
ATOM 3527 CA LEU B 51 -12 .460 26 .003 -3, .643 1 .00 22 .26 B C
ATOM 3528 CB LEU B 51 -11 .110 25 .407 -3, .219 1 .00 22 .70 B C
ATOM 3529 CG LEU B 51 -10 .067 25 .113 -4, .267 1 .00 25 .11 B C
ATOM 3530 CDl LEU B 51 -8 .762 24 .764 -3. .495 1 .00 25 .85 B C
ATOM 3531 CD2 LEU B 51 -10 .513 23 .968 -5, .183 1 .00 27 .79 B C
ATOM 3532 C LEU B 51 -13 .312 25 .997 -2, .406 1 .00 21 .80 B C
ATOM 3533 O LEU B 51 -13 .289 26 .962 -1, .646 1 .00 21 .46 B O
ATOM 3534 N PRO B 52 -14, .006 24 .886 -2, ,163 1 .00 21 .76 B N
ATOM 3535 CA PRO B 52 -14, .750 24 .709 -0, .921 1 .00 22 .13 B C
ATOM 3536 CB PRO B 52 -15, .340 23 .290 -1. .071 1 .00 23 .06 B C
ATOM 3537 CG PRO B 52 -15, .389 23 .059 -2. .525 1 .00 22 .20 B C
ATOM 3538 CD PRO B 52 -14, ,145 23 .724 -3. .058 1 .00 22 .25 B C
ATOM 3539 C PRO B 52 -13, .836 24 .809 0. .290 1 .00 22 .83 B C
ATOM 3540 O PRO B 52 -12, .682 24 .367 0. .252 1, .00 22 .05 B O
ATOM 3541 N VAL B 53 -14, .340 25 .400 1. .365 1, .00 23 .43 B N
ATOM 3542 CA VAL B 53 -13, ,579 25, .504 2. .581 1, ,00 23 .96 B C
ATOM 3543 CB VAL B 53 -14, .297 26, .368 3. .643 1, .00 24, .41 B C
ATOM 3544 CGI VAL B 53 -15, .583 25, .692 4. .134 1, .00 25, .13 B C
ATOM 3545 CG2 VAL B 53 -13, .360 26. .671 4. ,805 1, .00 25, .93 B C
ATOM 3546 C VAL B 53 -13, .324 24, .083 3. ,068 1, .00 24, .12 B C
ATOM 3547 O VAL B 53 -14, .153 23, .193 2. ,859 1, .00 24, .69 B O
ATOM 3548 N GLY B 54 -12, .158 23, .867 3. ,657 1, ,00 22 , .87 B N
ATOM 3549 CA GLY B 54 -11. .765 22 , .548 4. ,117 1, .00 22, .48 B C
ATOM 3550 C GLY B 54 -11. .067 21, ,662 3. ,092 1, .00 21, .22 B C
ATOM 3551 O GLY B 54 -10, .597 20, .606 3. .453 1, .00 21, ,63 B O
ATOM 3552 N THR B 55 -10. .977 22 , .091 1. ,837 1, .00 20. .24 B N
ATOM 3553 CA THR B 55 -10. .295 21, ,324 0. ,809 1 , ,00 19, .46 B C
ATOM 3554 CB THR B 55 -10. ,469 22. .006 -0. ,573 1, .00 19, .86 B C
ATOM 3555 OGl THR B 55 -11. .866 22 , ,158 -0. ,875 1, .00 22, .41 B O
ATOM 3556 CG2 THR B 55 -9. .957 21, .139 -1. ,701 1. .00 19, .94 B C
ATOM 3557 C THR B 55 -8. .788 21, .125 1. ,077 1. .00 17 , .93 B C
ATOM 3558 O THR B 55 -8. .057 22, .042 1. ,417 1. .00 16, .91 B O
ATOM 3559 N ILE B 56 -8. .336 19, .911 0. 849 1. ,00 17. .04 B N
ATOM 3560 CA ILE B 56 -6. .929 19. .571 0. 943 1. ,00 16. .44 B C
ATOM 3561 CB ILE B 56 -6. ,800 18, ,076 1. 232 1. ,00 15. .71 B C
ATOM 3562 CGI ILE B 56 -7. ,439 17. .782 2. 600 1. ,00 17. .41 B C
ATOM 3563 CDl ILE B 56 -7. ,353 16. .319 3. 098 1. ,00 17. .17 B C
ATOM 3564 CG2 ILE B 56 -5. ,347 17. .684 1. 247 1. ,00 16. .77 B C
ATOM 3565 C ILE B 56 -6. ,217 19. .981 -0. 336 1. ,00 16. .51 B C
ATOM 3566 O ILE B 56 -6. ,701 19. .691 -1. 434 1. ,00 17. ,03 B O
ATOM 3567 N VAL B 57 -5. .088 20, .678 -0, ,203 1, .00 15, .94 B N
ATOM 3568 CA VAL B 57 -4. .342 21, .200 -1. ,361 1, .00 16. .45 B C ATOM 3569 CB VAL B 57 -4.511 22.726 -1.488 1.00 16.21 B C
ATOM 3570 CGI VAL B 57 -6 .012 23 .092 -1 .672 1 .00 16 .73 B C
ATOM 3571 CG2 VAL B 57 -3 .991 23 .435 -0 .236 1 .00 18 .16 B C
ATOM 3572 C VAL B 57 -2 .853 20 .910 -1 .205 1 .00 16 .63 B C
ATOM 3573 O VAL B 57 -2 .393 20 .624 -0. .099 1 .00 16 .42 B O
ATOM 3574 N THR B 58 -2 .106 20 .982 -2. .299 1 .00 17 .06 B N
ATOM 3575 CA THR B 58 -0 .658 20 .801 -2 .247 1 .00 16 .63 B C
ATOM 3576 CB THR B 58 -0 .069 20 .712 -3 .654 1 .00 17 .18 B C
ATOM 3577 OGl THR B 58 -0 .660 21 .718 -4 .494 1 .00 15 .16 B O
ATOM 3578 CG2 THR B 58 -0 .423 19 .426 -4 .292 1 .00 19 .58 B C
ATOM 3579 C THR B 58 -0 .093 22 .017 -1 .536 1 .00 16 .72 B C
ATOM 3580 O THR B 58 -0 .756 23 .071 -1 .492 1 .00 16 .30 B O
ATOM 3581 N MET B 59 1 .103 21 .885 -0 .960 1 .00 16 .38 B N
ATOM 3582 CA MET B 59 1. .692 22 .982 -0, .180 1 .00 16 .07 B C
ATOM 3583 CB MET B 59 1 .960 22 .552 1 .254 1 .00 15 .92 B C
ATOM 3584 CG MET B 59 0 .668 22 .365 2 .012 1 .00 16 .14 B C
ATOM 3585 SD MET B 59 -0 .197 23 .961 2 .251 1 .00 17 .09 B S
ATOM 3586 CE MET B 59 -1 .612 23 .424 3. .218 1 .00 17 .23 B C
ATOM 3587 C MET B 59 2 .911 23 .607 -0 .816 1 .00 15 .83 B C
ATOM 3588 O MET B 59 3 .884 23 .983 -0 .134 1 .00 15 .99 B O
ATOM 3589 N GLU B 60 2 .837 23 .794 -2 .127 1 .00 15 .11 B N
ATOM 3590 CA GLU B 60 3 .838 24 .609 -2, .795 1 .00 15 .22 B C
ATOM 3591 CB GLU B 60 4 .155 24 .079 -4 .187 1 .00 15 .39 B C
ATOM 3592 CG GLU B 60 3 .299 24 .609 -5, .334 1 .00 15 .29 B C
ATOM 3593 CD GLU B 60 1 .845 24 .166 -5, .270 1 .00 19 .22 B C
ATOM 3594 OEl GLU B 60 1. .435 23 .486 -4, .308 1 .00 18 .05 B O
ATOM 3595 OE2 GLU B 60 1. .087 24 .526 -6, .195 1 .00 18 .12 B O
ATOM 3596 C GLU B 60 3, .344 26 .081 -2, .771 1 .00 15 .50 B C
ATOM 3597 O GLU B 60 2 .133 26 .356 -2. .798 1. .00 15 .17 B O
ATOM 3598 N TYR B 61 4, .292 27 .011 -2, .721 1 .00 16, .00 B N
ATOM 3599 CA TYR B 61 3, .982 28 .431 -2. .654 1 .00 17, .37 B C
ATOM 3600 CB TYR B 61 4, .938 29 .138 -1. .699 1. .00 17, .67 B C
ATOM 3601 CG TYR B 61 4, .671 30, .623 -1. .493 1. .00 18, .81 B C
ATOM 3602 CDl TYR B 61 5, ,474 31. .572 -2. .103 1, .00 21. .63 B C
ATOM 3603 CEl TYR B 61 5. .233 32. .895 -1. .943 1, .00 21. .96 B C
ATOM 3604 CZ TYR B 61 4, .207 33, .299 -1. ,134 1. .00 22 , .89 B C
ATOM 3605 OH TYR B 61 4, ,007 34, .644 -0. ,972 1, ,00 29. .91 B O
ATOM 3606 CE2 TYR B 61 3. .407 32, ,394 -0. ,507 1. .00 20. .81 B C
ATOM 3607 CD2 TYR B 61 3, .637 31, .059 -0. ,697 1. .00 19. .36 B C
ATOM 3608 C TYR B 61 4, .067 29, .037 -4. ,048 1, .00 18. .22 B C
ATOM 3609 O TYR B 61 5, .126 29, ,041 -4. ,654 1, .00 18 , .04 B 0
ATOM 3610 N ARG B 62 2, .943 29, .531 -4. ,564 1, .00 19 , .75 B N
ATOM 3611 CA ARG B 62 2 , .938 30. .226 -5. ,861 1. .00 21, .16 B C
ATOM 3612 CB ARG B 62 1. ,909 29, .625 -6. ,814 1, .00 21. .92 B C
ATOM 3613 CG ARG B 62 2, .189 28, .185 -7. ,196 1, ,00 25. .65 B C
ATOM 3614 CD ARG B 62 1. .385 27, .694 -8. ,421 1. .00 29, .39 B C
ATOM 3615 NE ARG B 62 1. ,516 26. .235 -8. ,639 1, .00 32. .04 B N
ATOM 3616 CZ ARG B 62 0. ,982 25. ,593 -9. .675 1 , ,00 33, .94 B C
ATOM 3617 NHl ARG B 62 1. ,129 24. .287 -9. ,798 1, .00 37. .42 B N
ATOM 3618 NH2 ARG B 62 0. ,292 26. .255 -10. ,586 1, .00 34. .66 B N
ATOM 3619 C ARG B 62 2. ,619 31, .687 -5. ,652 1, .00 21. ,86 B C
ATOM 3620 O ARG B 62 1. ,491 32. .033 -5. ,296 1. .00 21. ,34 B O
ATOM 3621 N ILE B 63 3. ,609 32. .547 -5. ,894 1. .00 23. ,01 B N
ATOM 3622 CA ILE B 63 3. ,503 33. ,950 -5. 502 1. .00 24. ,71 B C
ATOM 3623 CB ILE B 63 4. ,824 34. .707 -5. 709 1. .00 25. ,05 B C
ATOM 3624 CGI ILE B 63 4. ,790 36. .033 -4. 919 1. .00 28. ,83 B C
ATOM 3625 CDl ILE B 63 6. ,158 36. .609 -4. 546 1. .00 31. ,61 B C
ATOM 3626 CG2 ILE B 63 5. ,047 34. ,966 -7. 207 1. .00 26. ,58 B C
ATOM 3627 C ILE B 63 2. 367 34. ,699 -6. 216 1. ,00 24. ,34 B C
ATOM 3628 O ILE B 63 1. ,860 35. .675 -5. 672 1. ,00 25. ,45 B O
ATOM 3629 N ASP B 64 1. 961 34. .240 -7. 398 1. ,00 24. ,15 B N
ATOM 3630 CA ASP B 64 0. .901 34, .919 -8. .153 1, .00 24. ,63 B C
ATOM 3631 CB I 3ASP B 64 1. ,214 34, .889 -9. .664 0. .40 24. .94 B C ATOM 3632 CB .AASP B 64 1.201 34.873 -9.650 0.60 24.96 B C
ATOM 3633 CG : BASP B 64 0 .987 33 .514 -10 .307 0 .40 25 .65 B C
ATOM 3634 CG . AASP B 64 2, .403 35 .712 -10 .026 0 .60 25 .91 B C
ATOM 3635 OD1BASP B 64 0 .745 33 .465 -11 .536 0 .40 27 .17 B O
ATOM 3636 OD1AASP B 64 3 .136 35 .290 -10 .950 0 .60 27 .52 B O
ATOM 3637 OD2BASP B 64 1 .054 32 .426 -9 .686 0 .40 28 .49 B O
ATOM 3638 OD2AASP B 64 2 .704 36 .782 -9 .439 0 .60 26 .67 B O
ATOM 3639 C ASP B 64 -0 .514 34 .361 -7 .896 1 .00 24 .47 B C
ATOM 3640 O ASP B 64 -1 .515 34 .928 -8 .392 1 .00 24 .83 B O
ATOM 3641 N ARG B 65 -0 .601 33 .269 -7 .136 1 .00 21 .92 B N
ATOM 3642 CA ARG B 65 -1 .876 32 .615 -6 .893 1 .00 21 .12 B C
ATOM 3643 CB ARG B 65 -1 .677 31 .101 -6 .737 1 .00 19 .95 B C
ATOM 3644 CG ARG B 65 -2 .946 30 .305 -6 .463 1 .00 18 .99 B C
ATOM 3645 CD ARG B 65 -2 .730 28 .808 -6, .572 1 .00 19 .09 B C
ATOM 3646 NE ARG B 65 -1 .784 28 .369 -5, .554 1 .00 18 .66 B N
ATOM 3647 CZ ARG B 65 -1 .130 27 .214 -5 .534 1 .00 19 .41 B C
ATOM 3648 NHl ARG B 65 -1 .279 26 .300 -6, .470 1 .00 19 .74 B N
ATOM 3649 NH2 ARG B 65 -0 .311 26 .963 -4, .522 1. .00 21 .82 B N
ATOM 3650 C ARG B 65 -2 .556 33 .168 -5, .662 1, .00 20 .36 B C
ATOM 3651 O ARG B 65 -1, .896 33 .515 -4, .682 1, .00 21. .06 B O
ATOM 3652 N VAL B 66 -3 .875 33 .284 -5. .727 1, .00 20. .04 B N
ATOM 3653 CA VAL B 66 -4, .692 33 .540 -4, .560 1, .00 19. .79 B C
ATOM 3654 CB VAL B 66 -5, .266 34 .962 -4, .510 1, ,00 19. ,88 B C
ATOM 3655 CGI VAL B 66 -6, .036 35 .160 -3. .204 1, .00 21. .06 B C
ATOM 3656 CG2 VAL B 66 -4, .194 36 .004 -4. .607 1. .00 20, .96 B C
ATOM 3657 C VAL B 66 -5, .846 32 .525 -4. .551 1, .00 20, .19 B C
ATOM 3658 O VAL B 66 -6 .733 32 .512 -5, .444 1 .00 20 .12 B O
ATOM 3659 N ARG B 67 -5 .832 31 .654 -3, .557 1, .00 20 .01 B N
ATOM 3660 CA ARG B 67 -6 .916 30 .713 -3, .394 1, .00 20 .45 B C
ATOM 3661 CB ARG B 67 -6 .416 29 .437 -2, ,740 1, .00 20, .71 B C
ATOM 3662 CG ARG B 67 -5, .572 28 .538 -3. .626 1, .00 21, .28 B C
ATOM 3663 CD ARG B 67 -5 .471 27 .144 -3, .016 1, .00 23, .63 B C
ATOM 3664 NE ARG B 67 -4, .539 26 .204 -3, .632 1, .00 22, .50 B N
ATOM 3665 CZ ARG B 67 -3, .326 25 .921 -3. .160 1, .00 24, .19 B C
ATOM 3666 NHl ARG B 67 -2, .589 24 .992 -3. .759 1, .00 22, ,65 B N
ATOM 3667 NH2 ARG B 67 -2, .837 26, .571 -2. .106 1, .00 22, .06 B N
ATOM 3668 C ARG B 67 -8, .001 31. .368 -2. .543 1, .00 20. .49 B C
ATOM 3669 O ARG B 67 -7, .704 31. .990 -1. .529 1. .00 20. .17 B O
ATOM 3670 N LEU B 68 -9, ,255 31, .253 -2. .970 1. .00 20. .39 B N
ATOM 3671 CA LEU B 68 -10, .373 31, .705 -2. .148 1. .00 21. .05 B C
ATOM 3672 CB LEU B 68 -11. .323 32, .604 -2. .957 1. .00 21. .11 B C
ATOM 3673 CG LEU B 68 -10. .701 33, ,830 -3. .627 1. ,00 21, ,59 B C
ATOM 3674 CDl LEU B 68 -11. .745 34, .523 -4. .508 1. .00 22. .82 B C
ATOM 3675 CD2 LEU B 68 -10. ,187 34, .785 -2. .595 1. .00 22. .44 B C
ATOM 3676 C LEU B 68 -11. ,132 30, ,493 -1. ,648 1. .00 21. .12 B C
ATOM 3677 O LEU B 68 -11. .663 29, .709 -2. ,432 1. .00 21. .87 B O
ATOM 3678 N PHE B 69 -11. .209 30, .345 -0. ,345 1. ,00 21. .61 B N
ATOM 3679 CA PHE B 69 -11. .937 29, .226 0. .233 1. ,00 21. .97 B C
ATOM 3680 CB PHE B 69 -11. .151 28, .647 1. ,422 1. ,00 22. .17 B C
ATOM 3681 CG PHE B 69 -9, .896 27, .905 1. ,028 1. .00 19, .89 B C
ATOM 3682 CDl PHE B 69 -9, .938 26, .557 0. .737 1. .00 21, .41 B C
ATOM 3683 CEl PHE B 69 -8, .788 25, ,862 0. .370 1, .00 20, ,29 B C
ATOM 3684 CZ PHE B 69 -7. .598 26. .525 0. ,290 1, .00 21. .78 B C
ATOM 3685 CE2 PHE B 69 -7. .542 27, .879 0. .575 1. .00 21, .52 B C
ATOM 3686 CD2 PHE B 69 -8. ,691 28, ,557 0. ,950 1. .00 19, ,33 B C
ATOM 3687 C PHE B 69 -13. .321 29, .725 0. ,664 1. ,00 23. .11 B C
ATOM 3688 O PHE B 69 -13. .414 30, .651 1. .506 1. .00 22. .87 B O
ATOM 3689 N VAL B 70 -14. .371 29, .105 0. .111 1. .00 24 , .00 B N
ATOM 3690 CA VAL B 70 -15. .755 29, .578 0. ,289 1. .00 25, ,82 B C
ATOM 3691 CB VAL B 70 -16. .442 29, .958 -1. ,054 1, ,00 25, .72 B C
ATOM 3692 CGI VAL B 70 -15. .686 31, .063 -1. ,756 1. .00 26. .70 B C
ATOM 3693 CG2 VAL B 70 -16. .599 28, .756 -1. ,958 1. .00 26. .63 B C
ATOM 3694 C VAL B 70 -16. ,691 28, ,602 0. .996 1. .00 26. .63 B C ATOM 3695 O VAL B 70 -16.532 27.380 0,.899 1.00 27.14 B O
ATOM 3696 N ASP B 71 -17 .664 29 .155 1, .714 1 .00 27 .52 B N
ATOM 3697 CA ASP B 71 -18 .686 28 .352 2, .399 1 .00 29 .00 B C
ATOM 3698 CB ASP B 71 -19 .247 29 .132 3, .591 1 .00 28 .91 B C
ATOM 3699 CG ASP B 71 -20 .019 30 .392 3. .171 1 .00 28 .74 B C
ATOM 3700 ODl ASP B 71 -20 .161 31 .290 4, .018 1 .00 29 .94 B O
ATOM 3701 OD2 ASP B 71 -20 .509 30 .572 2, .032 1 .00 27 .69 B O
ATOM 3702 C ASP B 71 -19 .796 27 .931 1, ,413 1 .00 30 .37 B C
ATOM 3703 0 ASP B 71 -19 .646 28 .098 0, .217 1 .00 29 .90 B O
ATOM 3704 N LYS B 72 -20 .898 27 .376 1. .906 1 .00 33 .42 B N
ATOM 3705 CA LYS B 72 -21 .949 26 .822 1, .022 1, .00 34 .98 B C
ATOM 3706 CB LYS B 72 -22, .951 26 .004 1, .834 1, .00 35 .44 B C
ATOM 3707 CG LYS B 72 -22, .190 24 .925 2. .916 0, .00 40 .00 B C
ATOM 3708 CD LYS B 72 -22, .489 23 .435 2. .595 0. .00 40 .00 B C
ATOM 3709 CE LYS B 72 -21 .240 22 .528 2 .667 0 .00 40 .00 B C
ATOM 3710 NZ LYS B 72 -21 .120 21 .662 1, .456 0 .00 40 .00 B N
ATOM 3711 C LYS B 72 -22 .709 27 .904 0, .261 1 .00 36 .38 B C
ATOM 3712 O LYS B 72 -23 .332 27 .627 0, .766 1 .00 37 .77 B O
ATOM 3713 N LEU B 73 -22 .640 29 .138 0, .753 1 .00 37 .07 B N
ATOM 3714 CA LEU B 73 -23 .306 30 .286 0, .114 1 .00 37 .22 B C
ATOM 3715 CB LEU B 73 -23 .759 31 .265 1, .201 1 .00 37 .50 B C
ATOM 3716 CG LEU B 73 -24, .711 30 .678 2, .254 1, .00 40 .20 B C
ATOM 3717 CDl LEU B 73 -25, .387 31 .796 3. .053 1, .00 41 .53 B C
ATOM 3718 CD2 LEU B 73 -25, .782 29 .775 1, .612 1, .00 41 .89 B C
ATOM 3719 C LEU B 73 -22, .406 31 .008 0, .890 1, .00 36 .59 B C
ATOM 3720 O LEU B 73 -22, .781 32 .029 1. .482 1, .00 36 .18 B O
ATOM 3721 N ASP B 74 -21, .203 30 .468 1. .076 1, .00 36 .33 B N
ATOM 3722 CA ASP B 74 -20, .192 31 .084 1. .923 1, .00 35 .65 B C
ATOM 3723 CB ASP B 74 -20, .709 31 .252 3. .342 1, .00 36 .81 B C
ATOM 3724 CG ASP B 74 -20, .063 30 .283 4. .286 1, .00 40 .36 B C
ATOM 3725 ODl ASP B 74 -19. .429 30 .756 5. .259 1, .00 45 .75 B O
ATOM 3726 OD2 ASP B 74 -20. .108 29 .032 4. .101 1, ,00 45 .61 B O
ATOM 3727 C ASP B 74 -19, .646 32 .399 1. .381 1, .00 33 .94 B C
ATOM 3728 O ASP B 74 -19, .147 33 .249 2. .136 1, .00 34 .59 B O
ATOM 3729 N ASN B 75 -19, .696 32 .546 0. ,066 1, .00 31 .85 B N
ATOM 3730 CA ASN B 75 -19, .000 33 .634 0. ,597 1, .00 30 .66 B C
ATOM 3731 CB ASN B 75 -19, .877 34 .185 1. ,714 1, .00 30 .45 B C
ATOM 3732 CG ASN B 75 -21, .170 34 .793 1. ,176 1, .00 30 .77 B C
ATOM 3733 ODl ASN B 75 -21, .169 35 .401 0. , 102 1, .00 30 .57 B O
ATOM 3734 ND2 ASN B 75 -22, .275 34 .615 1. ,907 1, .00 30 .32 B N
ATOM 3735 C ASN B 75 -17, .628 33 .213 1. , 127 1, .00 29 .26 B C
ATOM 3736 O ASN B 75 -17, .433 32 .073 1. ,553 1, .00 29 .07 B O
ATOM 3737 N ILE B 76 -16, .685 34 .143 1. .086 1, .00 27 .83 B N
ATOM 3738 CA ILE B 76 -15, .337 33 .893 1. .540 1. .00 26 .88 B C
ATOM 3739 CB ILE B 76 -14, .511 35 .182 1. .455 1, .00 26 .51 B C
ATOM 3740 CGI ILE B 76 -14, ,474 35 .737 0. .020 1, .00 26 .61 B C
ATOM 3741 CDl ILE B 76 -14, .130 34 .728 1. ,039 1. .00 26 .87 B C
ATOM 3742 CG2 ILE B 76 -13, .137 34 .927 1. ,993 1, ,00 25 .62 B C
ATOM 3743 C ILE B 76 -15, .401 33, .382 2. .993 1, .00 26 .59 B C
ATOM 3744 O ILE B 76 -15, .994 34 .017 3, ,838 1, .00 26 .12 B O
ATOM 3745 N ALA B 77 -14 , .795 32, .237 3, ,263 1, .00 26 .58 B N
ATOM 3746 CA ALA B 77 -14 , .899 31, .582 4. ,576 1, .00 26 .79 B C
ATOM 3747 CB ALA B 77 -15. .129 30, .088 4. ,386 1, .00 26 .56 B C
ATOM 3748 C ALA B 77 -13. .670 31, .815 5. ,467 1, .00 26 .89 B C
ATOM 3749 O ALA B 77 -13, .764 31, .751 6. ,677 1, .00 27 .73 B O
ATOM 3750 N GLU B 78 -12, .518 32 , .072 4. ,871 1, .00 26 .32 B N
ATOM 3751 CA GLU B 78 -11. .355 32 , ,446 5. ,647 1. .00 26 .33 B C
ATOM 3752 CB GLU B 78 -10. .508 31, .216 6. ,041 1. .00 26 .80 B C
ATOM 3753 CG GLU B 78 -10. .106 30, .290 4. ,921 1, .00 27 .82 B C
ATOM 3754 CD GLU B 78 -9, .956 28, .827 5. .352 1, ,00 28 .92 B C
ATOM 3755 OEl GLU B 78 -9. ,785 28, .493 6. .559 1, .00 36 .40 B O
ATOM 3756 OE2 GLU B 78 -10 .001 27 .977 4. .472 1, .00 28 .50 B O
ATOM 3757 C GLU B 78 -10 .530 33 .478 4. .895 1, .00 25 .94 B C ATOM 3758 O GLU B 78 -10,.807 33.805 3.,712 1,.00 25.71 B O
ATOM 3759 N VAL B 79 -9, .527 33 .996 5, .594 1, .00 24 .85 B N
ATOM 3760 CA VAL B 79 -8, .712 35, .088 5. .102 1, .00 24 .88 B C
ATOM 3761 CB VAL B 79 -7, .692 35, .578 6. .167 1, .00 25 .77 B C
ATOM 3762 CGI . VAL B 79 -6, .814 36, .672 5. .583 1, .00 25 .38 B C
ATOM 3763 CG2 ! VAL B 79 -8, .396 36, .073 7. .456 1, .00 26 .10 B C
ATOM 3764 C VAL B 79 -7, .957 34, .652 3. .835 1, ,00 24 .07 B C
ATOM 3765 O VAL B 79 -7, .137 33, .742 3. .883 1, .00 23 .32 B O
ATOM 3766 N PRO B 80 -8, .275 35, .271 2. .699 1, .00 23 .27 B N
ATOM 3767 CA PRO B 80 -7, .533 35, .029 1. .466 1, .00 22 .93 B C
ATOM 3768 CB PRO B 80 -8, .296 35, .856 0. .426 1, ,00 23 .15 B C
ATOM 3769 CG PRO B 80 -9, .645 36, .042 1. .005 1. .00 23 .83 B C
ATOM 3770 CD PRO B 80 -9, .404 36, .194 2. .480 1, .00 23 .67 B C
ATOM 3771 C PRO B 80 -6, .101 35 .512 1. .601 1, .00 22 .05 B C
ATOM 3772 O PRO B 80 -5. .891 36 .588 2, .136 1 .00 20 .76 B O
ATOM 3773 N ARG B 81 -5, .147 34 .691 1, .190 1 .00 21 .54 B N
ATOM 3774 CA ARG B 81 -3 .765 35 .135 1, .074 1 .00 22 .93 B C
ATOM 3775 CB BARG B 81 -2. .937 34 .683 2, .298 0 .40 22 .99 B C
ATOM 3776 CB AARG B 81 -2 .865 34 .764 2. .275 0 .60 23 .27 B C
ATOM 3777 CG BARG B 81 -3 .684 34 .672 3, .645 0 .40 24 .27 B C
ATOM 3778 CG AARG B 81 -3 .278 33 .619 3, .175 0 .60 25 .79 B C
ATOM 3779 CD BARG B 81 -2, .996 33 .818 4, .744 0 .40 26 .79 B C
ATOM 3780 CD AARG B 81 -2, .615 33 .723 4, .575 0 .60 27 .67 B C
ATOM 3781 NE BARG B 81 -3 .975 33 .234 5, .664 0, .40 28 .96 B N
ATOM 3782 NE AARG B 81 -3 .283 32 .907 5, .581 0, .60 29 .38 B N
ATOM 3783 CZ BARG B 81 -4 .567 32 .053 5, .508 0, .40 29 .28 B C
ATOM 3784 CZ AARG B 81 -3, .934 33 .381 6, .642 0, .60 30 .17 B C
ATOM 3785 NH1BARG B 81 -4 .284 31. .274 4. .471 0, .40 31 .06 B N
ATOM 3786 NH1AARG B 81 -4, .020 34 .680 6, .867 0, .60 31 .08 B N
ATOM 3787 NH2BARG B 81 -5, .448 31 .645 6, .408 0, ,40 30 .62 B N
ATOM 3788 NH2AARG B 81 -4, .510 32 .545 7, .486 0, .60 31 .16 B N
ATOM 3789 C ARG B 81 -3, .115 34 .616 -0, , 178 1, .00 21 .97 B C
ATOM 3790 O ARG B 81 -3, .560 33 .632 -0, .761 1, ,00 21 .68 B O
ATOM 3791 N VAL B 82 -2, .029 35. .286 -0. .559 1, .00 20 .91 B N
ATOM 3792 CA VAL B 82 -1. .224 34 .884 -1, .679 1, .00 21 .03 B C
ATOM 3793 CB VAL B 82 -0. .126 35. .943 -1. .952 1, .00 21 .89 B C
ATOM 3794 CGI . VAL B 82 0, .869 35. .435 -2. .906 1, .00 22 .93 B C
ATOM 3795 CG2 ! VAL B 82 -0, .734 37, .243 -2. .483 1. .00 21 .44 B C
ATOM 3796 C VAL B 82 -0, .583 33, .532 -1. ,371 1, .00 20 .69 B C
ATOM 3797 O VAL B 82 -0. .235 33. .255 -0. .221 1, .00 19 .36 B O
ATOM 3798 N GLY B 83 -0, .469 32, .694 -2. .389 1. .00 19 .91 B N
ATOM 3799 CA GLY B 83 0, .382 31, .525 -2. ,331 1, .00 20 .16 B C
ATOM 3800 C GLY B 83 -0, .236 30, .295 -2. .955 1, ,00 20 .11 B C
ATOM 3801 O GLY B 83 -1, .416 30, ,328 -3. .319 1, .00 20 .36 B O
ATOM 3802 OXT GLY B 83 0, .468 29, .294 -3. , 104 1, .00 18 .79 B O

Claims

1. A method for constructing a variant of a parent subtilase, wherein the variant has at least one altered property as compared to said parent subtilase, which method comprises: a) analyzing the three-dimensional structure of the parent subtilase to identify, on the basis of an evaluation of structural considerations in relation to a JP170 three dimensional structure, at least one amino acid residue or at least one structural region of the subtilase, which is of relevance for altering said property; b) modifying the DNA of the polynucleotide encoding the parent to construct a polynucleotide encoding a variant subtilase, which in comparison to the parent subtilase, has been "modified by deletion, substitution or insertion of the amino acid residue or structural part identified in i) so as to alter said property; c) expressing the variant subtilase in a suitable host, and d) testing the resulting subtilase variant for said property.
2. A method of producing a subtilase variant, wherein the variant has at least one altered property as compared to a parent subtilase, which method comprises: a) producing a model structure of the parent subtilase on the three-dimensional structure of BPN', TY145 or JP170; or producing an actually determined three- dimensional structure of the parent subtilase, b) comparing the model or actual three-dimensional structure of the parent subtilase to the JP170 structure by superimposing the structures through matching the CA, CB, C, O, and N atoms of the active site residues, c) identifying on the basis of the comparison in step b) at least one structural part of the parent subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the n ucleic a cid sequence e ncoding the parent s ubtilase to p roduce a nucleic acid sequence encoding at least one deletion or substitution of one or more amino acids at a position corresponding to said structural part, or at least one insertion of one or more amino acid residues in positions corresponding to said structural part; e) performing steps c) and d) iteratively N times, where N is an integer with the value of one or more; f) preparing the variant resulting from steps a) - e); g) testing the properties of said variant; and h) optionally repeating steps a) - g) recursively; and i) selecting a subtilase variant having at least one altered property as compared to the parent subtilase. j) expressing the modified nucleic acid sequence in a host cell to produce the variant subtilase; k) isolating the produced subtilase variant;
I) purifying the isolated subtilase variant and m) recovering the purified subtilase variant.
3. The method of claims 1 or 2, wherein the parent subtilase belongs to the sub-group I-S1 , preferably selected from the group consisting of ABSS168, BASBPN, BSSDY, and
BLSCAR, or functional variants thereof having retained the characteristic of sub-group I- S1.
4. The method of claims 1 or 2, wherein the parent subtilase belongs to the sub-group I-S2, preferably selected from the group consisting of BLS147, BLS309, BAPB92, and
BYSYAB, or functional variants thereof having retained the characteristic of sub-group I- S2.
5. The method of claims 1 or 2, wherein the parent subtilase belongs to the TY145 type subgroup, preferably selected from the group comprising TY145, S39 and S41.
6. The method of claims 1 or 2, wherein the parent subtilase belongs to the JP170 type subgroup, preferably selected from the group comprising JP170, proteases KP43, KP1790, KP9860, Protease Ya, Protease E-1 and SD-521
7. The method of claim 6, wherein the parent JP170 type subtilase that is modelled is at least 58% homologous to SEQ ID NO:1 , preferably at least 60% homologous, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98% or even more preferably at least 99% homologous to the sequence of SEQ ID NO:1.
8. The method according to claims 6 or 7, wherein the parent JP170 subtilase is a JP170 like subtilase which is at least 58% h^mologous to the sequence of SEQ ID NO:1 , comprising the overall subtilisin fold and the following structural characteristics: a) a twisted beta-sheet with 7 strands, b) six alpha helices, c) at least three ion-binding sites, and not comprising the Strong and Weak ion-binding sites of the BPN' like subtilases, wherein the positions of said three ion-binding sites in the three-dimensional structure of the subtilase is defined by the distance to the c-alpha atoms of the three active site amino acid residues of the subtilases, that is Ser, His and Asp, and the c-alpha atom of the amino acid residue next to the active site Ser residue (next to Ser), wherein said distances between:
I) on-binding site 1 and
) Asp c-alpha atom is 26.70-28.70A, i) His c-alpha atom is 22.10-24.10A, ii) Ser c-alpha atom is 16.95-18.95A, v) next to Ser c-alpha atom is 15.30-17.3θA,
II) on-binding site 2 and
) Asp c-alpha atom is 33.50-35.50A, i) His c-alpha atom is 37-39A, ii) Ser c-alpha atom is 29.40-31.4θA, v) next to Ser c-alpha atom is 30.70-32.70A,
III) on-binding site 3 and
) Asp c-alpha atom is 41.50-43.50A, i) His c-alpha atom is 42.90-44.90A, ii) Ser c-alpha atom is 34.50-36.50A, v) next to Ser c-alpha atom is 35-37A.
9. The method of claim 1 or 2 for producing a JP170 type subtilase variant, wherein the variant has at least one altered property as compared to a parent subtilase, which method comprises: a) producing a model structure of the parent JP170 type subtilase on the three- dimensional structure of JP170; or producing an actually determined three- dimensional structure of the parent subtilase, b) comparing the model or actual three-dimensional structure of the parent JP170 type subtilase to the BPN' or TY145 structure by superimposing the structures through matching the CA, CB, C, O, and N atoms of the active site residues, c) identifying on the basis of the comparison in step b) at least one structural part of the parent JP170 type subtilase, wherein an alteration in said structural part is predicted to result in an altered property; d) modifying the nucleic acid sequence encoding the parent JP170 type subtilase to produce a nucleic acid sequence encoding at least one deletion or substitution of one or more amino acids at a position corresponding to said structural part, or at least one insertion of one or more amino acid residues in positions corresponding to said structural part; e) performing steps c) and d) iteratively N times, where N is an integer with the value
>_ of one or more; f) preparing the JP170 type subtilase variant resulting from steps a) - e); g) testing the properties of said variant; and h) optionally repeating steps a) - g) recursively; and i) selecting a JP170 type subtilase variant having at least one altered property as compared to the parent subtilase. j) expressing the modified nucleic acid sequence in a host cell to produce the variant subtilase; k) isolating the produced JP170 type subtilase variant;
I) purifying the isolated subtilase variant and m) recovering the purified subtilase variant.
10. The method of claim 9, wherein step (c) identifies amino acid residue positions located at a distance of 1θA or less to the ion-binding site of the JP170 type parent, preferably positions located at a distance of 6 A or less.
11. The method of claim 9, wherein step (c) identifies amino acid residue positions in the JP170 type parent, the modification of which provides for the removal of the ion binding site by modification of at least one of the positions identified.
12. The method of claim 9, wherein step (c) identifies amino acid residue positions in highly mobile regions of the JP170 type parent.
13. The method of claim 9, wherein step (c) identifies amino acid residue positions in mobile regions of the JP170 type parent.
14. The method of claim 9, wherein step (c) identifies amino acid residue positions in the parent JP170 type, the modification of which may create at least one disulfide bridge by insertion of or substitution with at lease one Cys residue.
15. The method of claim 9, wherein steps (c) and (d) provide for constructing a variant of a parent JP170 type having a modified surface charge distribution by: c') identifying, on the surface of the parent JP170 type, at least one charged amino acid residue; d') modifying the charged residue identified in step (a) through deletion or substitution with an uncharged amino acid residue;
16. The method of claim 9, wherein steps (c) and (d) provide for constructing a variant of a parent JP170 type having a modified surface charge distribution by: c") identifying, on the surface of the parent JP170 type, at least one position being occupied by an uncharged amino acid residue; d") modifying the charge in that position by substituting the uncharged amino acid residue with a charged amino acid residue or by insertion of a charged amino acid residue at the position.
17. The method of claim 9, wherein steps (c) and (d) provide for constructing a variant of a parent JP170 type having a modified surface charge distribution by: c"') identifying, on the surface of the parent JP170 type, at least one charged amino acid residue; d'") substituting the charged amino acid residue identified in step (a) with an amino acid residue having an opposite charge.
18. The method of claim 9, wherein step (c) identifies amino acid residue positions in the parent JP170 type, the modification of which to Pro may create a JP170 type variant exhibiting improved stability.
19. The method of claim 9, wherein step (c) identifies amino acid residue positions in the parent JP170 type at a distance of less than 1θA from the active site residues.
20. The method of one or more of claims 9 to 19, wherein N in step (e) is an integer between 1 and 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, or 2.
21. A J P170 type s ubtilase variant comprising at l east one m odification i n a n a mino acid residue in a position located at a distance of 1θA or less to the ion-binding site, preferably positions located at a distance of 6 A or less.
22. The variant of claim 21 , wherein modifications are made in at least one of the positions:: a) ion-binding site 1 : 183, 184, 185, 186, 187, 188, 189, 191 , 193, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 224 and 225, b) ion-binding site 2: 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392 and 393, c) ion-binding site 3: 348, 350, 352, 363, 364, 365, 366, 367, 369, 370, 380, 381 , 382, 383, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 414, 415, 416, 417, 418, 419, 420, preferably the modifications: S193Q.Y; H200D.N; H200D.N+D196N; N390D; N391 D; G394N,Q,F,Y,S and W392S,N,Q.
23. A JP170 type subtilase variant comprising the introduction of a ion-binding site corresponding to the Strong ion-binding site of the BPN' like family subtilases, wherein said variant has a partial or full deletion of the region N79-N82 of SEQ ID NO:1 and subsequent insertion of one or more amino acid residues, preferably insertion of the sequence LNNSIQV (SEQ ID NO:5) followed by the substitution A45D.N and optionally the substitutions E44P.T and/or R47Q.
24. A JP170 type subtilase variant inϋ which one or more ion-binding sites have been removed, wherein said variant comprises partial or full deletion of the region N186-N199 of SEQ ID NO:1 and subsequent insertion of one or more amino acid residues, preferably insertion of the sequence SSN (SEQ ID NO:6), and preferably further comprising one or both of the substitutions I7Q and V3Y.
25. A BPN' type subtilase variant in which the ion-binding sites has been removed, wherein said variant comprises: a) partial or full deletion of the region A194-L196 (Savinase in BPN' numbering) or a corresponding region in another BPN' like subtilase and insertion of three or more amino acid residues, preferably insertion of P209-P217 from JP170 or a corresponding region in another JP170 like subtilase and partial or full deletion of the region L75-L82 (Savinase in BPN' numbering) or a corresponding region in said other BPN' like subtilase and insertion of one or more amino acid residues, preferably insertion of H83-, 122! from TY145 or a corresponding region in o er u i ase or b) partial or full deletion of the region A194-L196 (Savinase in BPN" numbering) or a corresponding region in another BPN' like subtilase and insertion of three or more amino acid residues, preferably insertion of P209-P217 from JP170 or a corresponding region in another JP170 like subtilase and partial or full deletion of the L75-L82 (Savinase in BPN' numbering) or a o corresponding region in said other BPN' like subtilase and insertion of one or more amino acid residues, preferably insertion of N79-K83 from JP170 or a corresponding region in another JP170 like subtilase.
26. A JP170 type subtilase variant comprising at least one modification in a position selected from the group comprising positions:
13, 14, 15, 16, 17, 18, 37, 38, 39, 40, 41 , 42, 43, 47, 48, 49, 50, 58, 59, 60, 67, 96, 97, 98, 99, 107, 108, 109, 110, 111 , 131 , 132, 133, 134, 152, 153, 163, 164, 165, 166, 188, 189, 190, 191 , 193, 195, 210, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 326, 327, 328, 329, 330, 331 , 332, 337, 338, 339, 340, 342, 355, 356, 357, 359, 360, 372, 373, 374, 375, 376, 377, 378, 3&4, 385, 387, 388, 389, 390, 391 , 392, 404, 405, 406, 407, 408, 409, 410, 411 and 419.
27. The subtilase variant according to claim 26 comprising one or more of the modifications: W240H.Y; G355A.S; S356T.N; T357N,Q,D,E,P; A359S,T,N,Q or S360T.N.
28. A variant subtilase comprising an alteration in one or more positions which are within a distance of 1θA from a CI2 inhibitor which is bound to the active site of JP170, wherein the positions, as specified in SEQ ID NO:1 are:
29, 30, 31 , 32, 64, 67, 68, 69, 70, 71 , 72, 93, 96, 97, 98, 107, 108, 109, 110, 113, 114, 127, 128, 129, 130, 131 , 132, 133, 134, 136, 138, 139, 140, 141 , 144, 157, 174, 180, 181 , 182, 183, 191 , 193, 202, 203, 205, 206, 207, 211 , 223, 224, 225, 226, 234, 235, 236, 237, 238, 239, 240, 241 , 249, 250, 251 , 252, 253, 254, 257, 258, preferably comprising the substitution W129L.
o
29. A JP170 like subtilase variant comprising one or more disulfide bridges introduced by one or more of the following modifications: G21C+A86C, V26C+A265C, G57C+G105C, G74C+A229C, Q111 C+N143C, G160C+S170C, A286C+V349C, A27C+A122C, A45C+G78C, V72C+P258C, G78C+A229C, D98C+G104C, Q111C+Y147C, G135C+G167C, R142C+P354C, V144C+A178C, G182C+P217C, A183C+G223C, A195C+Y225C, F271 C+P279C, A287C+A430C, A293C+T310C, E322C+S428C, S324C+A332C, S327C+P424C, D352C+N397C, G355C+T362C, G291C+S314C, Preferably one or more of the substitutions: G21C+A86C, V26C+A265C, G57C+G105C, G74C+A229C, Q111C+Y143C, G160C+S170C, A286C+V349C, A4C+P222C and A27C+A117C wherein the positions correspond to the positions in SEQ ID NO:1.
30. A JP170 type subtilase variant comprising an alteration in one or more of the positions N79, N316, L381 , K9, and K313, preferably comprising one or more of the substitutions N79D, N316D, L381 D, K9R, and K313R of SEQ ID NO:1.
31. A JP170 type subtilase variant comprising an alteration in one or more of the positions 22, 44, 110, 139, 140, 166, 198, 201 , 203, 231 , 282, 356, 357 and 378, preferably comprising one or more of the substitutions: Q22P, E44P, L110P, T139P, D140P, S166P I198P, V201 P, Q203P, S231P, S282P, S356P, T357P and K378P.
32. A JP170 like subtilase variant comprising a deletion of the region 311-433, preferably deletion of positions 317-433 or 315-433, further comprising one or more of the substitutions L283N.Q; A290S.N and W306H,Y,K.
33. An isolated nucleic acid sequence comprising a nucleic acid sequence, which encodes for the subtilase variant defined or produced in any of claims 21 to 32.
34. An isolated nucleic acid sequence according to claim 33, wherein the nucleic acid sequence is selected form the group consisting of: a) a nucleic acid sequence encoding an enzyme having at least 58% homology with the amino acid sequence shown in SEQ ID NO:1 , and b) a nucleic acid sequence which hybridizes under low stringency conditions, preferably under medium stringency conditions, in particular under high stringency conditions, with a complementary strand of the nucleic acid sequence encoding an enzyme having at least 58% homology with the amino acid sequence shown in SEQ ID NO:1 , or c) a subsequence of any of a) or b) of at least 100 nucleotides.
35. An isolated nucleic acid construct comprising a nucleic acid sequence as defined in any of claims 33 or 34, operably linked to one or more control sequences capable of directing the expression of the polypeptide in a suitable expression host.
36. A recombinant host cell comprisinci24e nucleic acid construct of claim 35.
37. A method for producing the variant defined in any of claims 21-32, the method comprising: a) cultivating the recombinant host cell of claim 36 under conditions conducive to the production of the subtilase variant; and b) recovering the variant.
38. A detergent composition comprising a JP170 type subtilase variant or a BPN' type subtilase variant of any of claims 21-32.
39. Use of a JP170 type subtilase variant or a BPN' type subtilase variant of claims 21- 32 in cleaning or washing applications.
EP04722236A 2003-03-21 2004-03-22 Subtilases Ceased EP1608744A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP09152523A EP2163616A3 (en) 2003-03-21 2004-03-22 Jp170 subtilase variants

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DK200300435 2003-03-21
DKPA200300435 2003-03-21
PCT/DK2004/000194 WO2004083362A2 (en) 2003-03-21 2004-03-22 Modification of subtilases using protein modelling based on the jp170 three-dimensional structure

Publications (1)

Publication Number Publication Date
EP1608744A2 true EP1608744A2 (en) 2005-12-28

Family

ID=33016787

Family Applications (2)

Application Number Title Priority Date Filing Date
EP04722236A Ceased EP1608744A2 (en) 2003-03-21 2004-03-22 Subtilases
EP09152523A Withdrawn EP2163616A3 (en) 2003-03-21 2004-03-22 Jp170 subtilase variants

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP09152523A Withdrawn EP2163616A3 (en) 2003-03-21 2004-03-22 Jp170 subtilase variants

Country Status (5)

Country Link
EP (2) EP1608744A2 (en)
JP (2) JP4908196B2 (en)
CN (2) CN101851615B (en)
AR (1) AR043746A1 (en)
WO (1) WO2004083362A2 (en)

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7294499B2 (en) 2003-01-30 2007-11-13 Novozymes A/S Subtilases
ATE533843T1 (en) 2004-09-21 2011-12-15 Novozymes As SUBTILATE
US7816115B2 (en) 2004-09-21 2010-10-19 Novozymes A/S Subtilases
US7473544B2 (en) * 2004-10-08 2009-01-06 Kao Corporation Alkaline protease
DE102007032111B4 (en) * 2007-07-09 2017-07-20 Henkel Ag & Co. Kgaa New proteases and detergents and cleaning agents containing these proteases
JP6067409B2 (en) 2012-04-10 2017-01-25 花王株式会社 Method for improving solubility of alkaline protease
EP2832853A1 (en) * 2013-07-29 2015-02-04 Henkel AG&Co. KGAA Detergent composition comprising protease variants
EP3212785A2 (en) * 2014-10-30 2017-09-06 Novozymes A/S Protease variants and polynucleotides encoding same
PL3608403T3 (en) * 2014-12-15 2025-06-23 Henkel Ag & Co. Kgaa Detergent composition comprising subtilase variants
CN118497177A (en) * 2014-12-19 2024-08-16 诺维信公司 Protease variants and polynucleotides encoding the same
CN117904083A (en) 2014-12-19 2024-04-19 诺维信公司 Protease variants and polynucleotides encoding the same
WO2016097357A1 (en) 2014-12-19 2016-06-23 Novozymes A/S Protease variants and polynucleotides encoding same
US10590367B2 (en) 2014-12-19 2020-03-17 Novozymes A/S Protease variants and polynucleotides encoding same
CN111615559B (en) 2018-01-16 2024-01-16 花王株式会社 Evaluation method of keratin stain cleansers and keratin stain decomposing ability
US11760793B2 (en) 2020-12-18 2023-09-19 University Of Tennessee Research Foundation Antibody for skewing sex ratio and methods of use thereof

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK386586D0 (en) * 1986-08-14 1986-08-14 Novo Industri As PROTEASE, PROCEDURE FOR PREPARING IT AND USING THEREOF
US5567601A (en) * 1993-06-01 1996-10-22 University Of Maryland Subtilisin mutants lacking a primary calcium binding site
EP0724629A1 (en) * 1993-10-18 1996-08-07 Degussa Aktiengesellschaft Method for controlled modification of enzymes, enzymes modified in this way and the use of such enzymes
ES2390901T3 (en) * 1995-02-03 2012-11-19 Novozymes A/S Method for designing alpha-amylase mutants with predetermined properties
US5891701A (en) * 1997-06-12 1999-04-06 Novo Nordisk Biotech Inc. Nucleic acids encoding a polypeptide having protease activity
AU776534B2 (en) * 1998-10-30 2004-09-16 Novozymes A/S Low allergenic protein variants
CN100482790C (en) * 2000-03-08 2009-04-29 诺维信公司 Variants with altered properties
US6803222B2 (en) * 2000-11-22 2004-10-12 Kao Corporation Alkaline proteases
JP4143274B2 (en) * 2001-04-12 2008-09-03 花王株式会社 Alkaline protease
JP2002218989A (en) * 2000-11-22 2002-08-06 Kao Corp Alkaline protease

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004083362A2 *

Also Published As

Publication number Publication date
CN1791672A (en) 2006-06-21
JP2006521795A (en) 2006-09-28
CN101851615A (en) 2010-10-06
EP2163616A3 (en) 2010-05-26
JP2012045000A (en) 2012-03-08
EP2163616A2 (en) 2010-03-17
WO2004083362A2 (en) 2004-09-30
WO2004083362A3 (en) 2004-11-04
JP4908196B2 (en) 2012-04-04
CN101851615B (en) 2014-08-06
AR043746A1 (en) 2005-08-10

Similar Documents

Publication Publication Date Title
US8563289B2 (en) Protease variants
US10144922B2 (en) Subtilase variants and compositions comprising the same
US8008057B2 (en) Subtilases
WO2004067737A2 (en) Subtilases
WO2004083362A2 (en) Modification of subtilases using protein modelling based on the jp170 three-dimensional structure
CA2200563C (en) Calcium free subtilisin mutants
WO2014194117A2 (en) Novel metalloproteases
US6541234B1 (en) Calcium free subtilisin mutants
CN100415880C (en) I-S1 and I-S2 subgroup subtilases having at least one additional amino acid residue between positions 97 and 98
EP1914307A2 (en) Subtilases
EP1183342A1 (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additional amino acid residue between positions 128 and 129
EP1183339A1 (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having at least one additional amino acid residue between positions 129 and 130

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20051021

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20060425

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 20090519