WO2013041223A1

WO2013041223A1 - Crystal structure of the 60s eukaryotic ribosomal subunit and its uses

Info

Publication number: WO2013041223A1
Application number: PCT/EP2012/003924
Authority: WO
Inventors: Nenad Ban; Sebastian KLINGE; Felix VOIGTS-HOFFMANN; Marc LEIBUNDGUT
Original assignee: Eth Zurich
Priority date: 2011-09-20
Filing date: 2012-09-20
Publication date: 2013-03-28

Abstract

The present invention is directed to a crystal of the eukaryotic 60S ribosomal subunit of Tetrahymena thermophila in complex with the eukaryotic initiation factor 6 (elF6) of Tetrahymena thermophila, optionally together with a ligand, preferably an antibiotic such as cycloheximide; a method of producing said crystal and a composition comprising said crystal. In addition, the present invention relates to a computer system and a machine- readable medium comprising the atomic coordinates of the Tetrahymena thermophila 60S ribosomal subunit or coordinates derived therefrom as well as the use of said atomic coordinates for designing and selecting ligands that inhibit or modulate protein synthesis in eukaryotes. Also, the invention provides a method for identifying a potential ligand, preferably an inhibitor or modulator of a eukaryotic 60S ribosomal subunit.

Description

Crystal structure of the 60S eukaryotic ribosomal subunit and its uses

The present invention is directed to a crystal of the eukaryotic 60S ribosomal subunit of Tetrahymena thermophila in complex with the eukaryotic initiation factor 6 (elF6) of Tetrahymena thermophila, optionally together with a ligand, preferably an antibiotic such as cycloheximide; a method of producing said crystal and a composition comprising said crystal. In addition, the present invention relates to a computer system and a machine-readable medium comprising the atomic coordinates of the Tetrahymena thermophila 60S ribosomal subunit or coordinates derived therefrom as well as the use of said atomic coordinates for designing and selecting ligands that inhibit or modulate protein synthesis in eukaryotes. Also, the invention provides a method for identifying a potential ligand, preferably an inhibitor or modulator of a eukaryotic 60S ribosomal subunit.

Background of the invention

Ribosomes catalyze protein synthesis in all domains of life. These ribonucleo- protein particles consist of two subunits with distinct functions. mRNA information is decoded by the small ribosomal subunit and peptide bond formation is mediated by the RNA component of the large ribosomal subunit. Ribosome-associated factors bind to the large ribosomal subunit and interact with nascent polypeptides emerging from the ribosomal tunnel. The large subunit is also a target for antibiotics that interfere with the peptidyl transferase reaction and with the progression of the nascent polypeptide chain through the tunnel.

Prokaryotic translation has been investigated for over a decade using the crystal structures of the small (30S) and large (50S) ribosomal subunits as well as the complete (70S) prokaryotic ribosome. In recent years, structures of prokaryotic ribosomes in complex with protein factors involved in translation have provided insights in the various stages of protein synthesis at atomic resolution.

However, the molecular understanding of protein synthesis in eukaryotes is only now emerging. Eukaryotic ribosomes are considerably larger than their bacterial counterparts. Both eukaryotic ribosomal subunits contain numerous RNA expansion segments (ESs), which co-evolved with many additional eukaryotic-specific ribosomal protein elements. As a consequence, the eukaryotic 60S subunit in yeast or T. thermophila has a total molecular weight of approximately 2 MDa compared to 1.3 MDa of the 50S subunit in Escherichia coli.

The increased level of structural complexity of eukaryotic ribosomes reflects functional differences between prokaryotes and eukaryotes. First, ribosome biogenesis in eukaryotic cells is elaborate. It takes place in different cellular compartments and involves approximately 200 trans-acting proteins in the processing and modification of ribosomal RNA, the import of ribosomal proteins into the nucleus as well as the export of pre-ribosomal subunits into the cytoplasm. Second, the regulation of protein synthesis, which mostly focuses around the eukaryotic 40S ribosomal subunit, is much more complex in eukaryotes.

Recently, cryo-electron microscopy (EM) reconstructions of yeast and wheat germ 80S ribosomes at 5.5-6.1 A resolution and a partial interpretation of crystallographic data from the 80S yeast ribosome at 4.15 A have provided for the positions and topology of eukaryotic RNA expansion segments and several proteins, for which homologous structures are known. However, these studies did not allow to correctly assign and build many eukaryotic proteins. The recent crystal structure of the T. thermophila small ribosomal subunit (40S) in complex with elF1 represents the first complete atomic model of the eukaryotic 40S ribosomal subunit (see Rabl et al., Science 331 , 730, 201 1 ), whereas a corresponding structure of the large ribosomal subunit (60S) has so far remained elusive.

The 60S ribosomal subunit is subject to several regulatory processes during initiation. Binding of eukaryotic initiation factor 6 (elF6) to the large ribosomal subunit inhibits subunit joining and thus prevents translation initiation (see Ceci et al., Nature 426, 579, 2003). The crystal structures of isolated elF6 and its archaeal homologue alF6 in isolation have highlighted its pentameric shape. The mechanism by which elF6 prevents 80S complex formation is most likely by steric hindrance, however, the exact interaction between elF6 (alF6) and the large ribosomal subunit is currently unclear, as chemical probing and low-resolution EM data have provided conflicting evidence (Benelli et al., Nucleic Acids Res. 37, 256, 2009; Gartmann et al. J. Biol. Chem. 285, 14848, 2010). In addition to its function as anti-association factor, elF6 has also been implicated in 60S maturation.

It is the object of the present invention to provide a crystal of the eukaryotic 60S ribosomal subunit, the atomic coordinates of which are useful for evaluating function and regulation of the subunit as well as for designing and identifying potential ligands and protein synthesis inhibitors. This problem is solved by providing a crystal of the eukaryotic 60S ribosomal subunit of Tetrahymena thermophila in complex with the eukaryotic initiation factor 6 (elF6) of Tetrahymena thermophila, comprising an average thickness greater than 10 pm and diffracting X-rays to a resolution of at least 7.0, preferably 3.5 A.

The crystal of the present invention is the first crystalline structure of a eukaryotic 60S ribosomal subunit ever to be provided. As used herein, the term "crystal" refers to any three-dimensional ordered array of molecules that diffracts X-rays. The term

"average thickness" of a crystal describes the size of the crystal measured along the principal axis of the parallelepiped shaped block, where the molecules form a three- dimensional array. In the instant case, the average size of the three axis is greater than 10 μιτι, i.e. (x+y+z)/3.

In a preferred embodiment, the crystal of the invention is characterized by having a monoclinic space group P2i with unit cell dimensions of a=320.5±10 A, b=289±10 A, c=536±10 A and with a β angle of 9 .5±0.5 °. The term "monoclinic space group Ρ2 refers to the arrangement of symmetry elements of a crystal as defined in the International Tables for Crystallography, Volume A, Space Group Symmetry, edited by Th. Hahn, Kluwer Academic Publishers, 2002.

In a further preferred embodiment of the invention, the structure of the crystal of the invention is defined by the coordinates deposited under Protein Data Bank accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1 C and 4A1D for molecule 4 of the asymmetric unit.

The above coordinates were deposited with the RCSB Protein Data Bank before the filing of the priority patent application, i.e. on September 14, 2011 , and are publicly available without any restrictions for electronic retrieval under http://www.rcsb.org/pdb/- home/home.do. The deposited coordinates referred herein are incorporated in toto by reference. The term "asymmetric unit" as used above refers to a minimal set of atomic co-ordinates that when operated upon by the symmetry operations of a crystal will regenerate the entire crystal.

Preferably, the crystal of the invention consists of the eukaryotic 60S ribosomal subunit in complex with eukaryotic initiation factor 6 comprising a ligand, preferably in complex with the eukaryotic 60S ribosomal subunit and/or the eukaryotic initiation factor as defined by the coordinates deposited under Protein Data Bank accession numbers (i) 4A1 E and 4A18 for molecule , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1 A and 4A1 B for molecule 3 and (iv) 4A1C and 4A D for molecule 4 of the asymmetric unit.

In a preferred embodiment the crystal of the invention consists of the eukaryotic 60S ribosomal subunit in complex with eukaryotic initiation factor 6, preferably in complex with a ligand, preferably located or disposed therein, more preferably comprising a ligand, preferably in complex with the eukaryotic 60S ribosomal subunit and/or the eukaryotic initiation factor as defined by the coordinates deposited under Protein Data Bank accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1C and 4A1 D for molecule 4 of the asymmetric unit.

The term "ligand" as used herein, refers to any substance binding or otherwise interacting with a eukaryotic 60S ribosomal subunit, preferably the 60S ribosomal subunit of Tetrahymena thermophila, more preferably in complex with the eukaryotic initiation factor 6 (elF6) of Tetrahymena thermophila, and includes substances binding or otherwise interacting with functional fragments of the 60S ribosomal subunit. Ligands for use in the invention include single atoms, in particular heavy atoms, typically heavy metal ions, ribosomal antibiotics, tRNA, peptidyl tRNA, amino acyl tRNA, signal recognition particles ("SRP") or derivatives thereof that maintain the capacity to bind or interact otherwise with the 60S ribosomal subunit.

In a preferred embodiment, the ligand comprised by the crystal of the invention is an inhibitor or modulator of protein synthesis, preferably an antibiotic, more preferably selected from the group consisting of chloramphenicols, macrolides, lincosamides, streptogramins, althiomycins, oxazolidinones, nucleotide analogs, thiostreptons

(including the micrococcin family), peptide antibiotics, glutarimides, trichothecenes, TAN -I OS7, pleuromutilins, hygromycins, betacins, eveminomicins, boxazomycins and fusidanes, most preferably selected from the group consisting of puromycin, cyclo- heximide and chloramphenicol.

In a further preferred embodiment the crystal of the invention has an average thickness of 10 to 600 pm, preferably an average thickness selected from the group consisting of 10 to 50, 51 to 100, 101 to 150, 151 to 200, 100 to 300 and 300 to 600 pm.

Furthermore, it is preferred that the crystal of the invention is one wherein the eukaryotic 60S ribosomal subunit in the crystal is homologous to the eukaryotic 60S ribosomal subunit from Tetrahymena thermophila, and the similarity, preferably identity is at least 30%, more preferably at least 70 or 80, most preferably 90, 95 or 99 % identity to the Tetrahymena thermophila ribosomal RNA and ribosomal proteins that they have in common.

As used herein, the term "homologous" is understood to indicate amino acid and/or nucleic acid sequence similarity or identity to a specified amino acid and/or nucleic acid sequence. For determining the sequence identity among polypeptides, the skilled person can revert to a number of standard algorithms known to those of skill in the art. Preferably, the BLAST programs at http://www.expasy.org/tools/blast/ and http://www.ncbi.nlm.- nih.gov/BLAST/Blast.cgi?CMD=Web&LAYOUT=TwoWindows&AUTO_FORMAT=Semia uto5^LIGNMENTS=250&ALIGNMENT_VIEW=Pairwise&CDD_SEARCH=on&CLIENT= web&DATABASE=nr&DESCRIPTIONS=500&ENTREZ_QUERY=%28none%29&EXPEC T=10&FILTER=L&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&l_THRESH =0.005&MATRIX_NAME=BLOSUM62&NCBI_GI=on&PAGE=Proteins&PROGRAM=blas tp&SERVICE=plain&SET_DEFAULTS.x=41 &SET_DEFAULTS.y=5&SHOW_OVERVIE W=on&END_OF_HTTPGET=Yes&SHOW_LINKOUT=yes&GET_SEQUENCE=yes, more preferably with the default settings, are used to identify the amino acid sequence identity of a protein, protein fragment or protein derivative of the present invention.

The identity of related nucleic acid molecules can be determined with well-known methods. In general, special computer programs are employed that use algorithms adapted to accommodate the specific needs of this task. Preferred methods for determining identity begin with the generation of the largest degree of identity among the sequences to be compared. Preferred computer programs for determining the identity among two nucleic acid sequences comprise, but are not limited to, BLASTN (Altschul et al., J. Mol. Biol., 215, 403-410,1990) and LALIGN (Huang and Miller, Adv. Appl. Math., 12, 337-357, 1991 ). The BLAST programs can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST handbook, Altschul et al., NCB NLM NIH Bethesda, MD 20894).

In another preferred aspect, the present invention relates to a method of producing a crystal of the invention.

Preferably the method is one, comprising the steps of:

(i) mixing the eukaryotic 60S ribosomal subunit from Tetrahymena thermophila and the eukaryotic initiation factor 6 (elF6) of Tetrahymena thermophila under conditions that allow for complex formation,

(ii) crystallizing the ribosomal subunit and the initiation factor in an aqueous solution comprising

(a) 50 to 200, preferably 75 to 150, more preferably about 100 mM Bis-Tris

propane, preferably Bis-Tris propane HCI,

(b) 50 to 200, preferably 75 to 150, more preferably about 100 mM acetate,

preferably potassium acetate,

(c) 3 to 10, preferably 5 to 7, more preferably about 6 % by weight PEG

(polyethylene glycol), preferably PEG 20K (e.g. from Sigma Aldrich) at a temperature of 15 to 30, preferably 18 to 25, more preferably about 20 °C, and a pH of 5.5 to 7.5, preferably 6 to 7, more preferably about 6.5.

In a preferred embodiment, the crystallization method of the invention comprises further growth crystallization comprising the steps:

(iii) mixing about equal aqueous amounts of the 60S ribosomal subunit elF6 complex with

(ai) 50 to 200, preferably 75 to 150, more preferably about 100 mM Bis-Tris propane, preferably Bis-Tris propane HCI,

(aii) 50 to 200, preferably 75 to 150, more preferably about 100 mM KCI,

(aiii) 4.5 to 7, preferably 5.4 to 6.0 % by weight PEG, preferably PEG 10K to 20K, more preferably 14K (e.g. from Sigma Aldrich),

(aiv) 1 to 5, preferably 2 to 4, more preferably about 3 mM spermidine,

preferably by the sitting drop vapor diffusion method and at a pH of 6 to 7.5, preferably 6.5 to 7, more preferably about 6.7, and optionally adding 0.5 to 2, preferably about 1 mM of a ribosomal ligand, preferably an antibiotic, preferably cycloheximide during growth crystallization.

Preferably growth crystallization is for 1 to 10, preferably 3 to 7, more preferably about 5 days.

The addition of a ribosomal ligand, preferably an antibiotic, preferably

cycloheximide during crystallization will result in morphologically improved crystals of larger size with better diffraction properties than without the ligand.

For dehydration and cryoprotection of the crystals of the invention comprising the 60S ribosomal subunit and the initiation factor 6 and optionally a ligand, preferably an antibiotic, more preferably cycloheximide, stepwise addition of PEG, preferably PEG 14K (10 to 20, preferably about 5 % by weight) and ethylene glycol (20 to 30, preferably about 25 % by weight) as cryoprotectant is preferred, optionally followed by introducing the crystal into liquid nitrogen.

In a preferred embodiment, the present invention relates to a crystal produced according to the above method of the invention.

In a further preferred aspect, the present invention is directed to a composition, preferably a composition of matter, comprising a crystal according to the invention and/or a crystal produced according to the invention.

Another aspect of the invention is a computer system comprising:

(A) a memory storing data defining one or more target regions

(a1 ) by atomic coordinates of the Tetrahymena thermophila 60S ribosomal subunit

deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1 C and 4A1 D for molecule 4 of the asymmetric unit, and/or

(a2) by atomic coordinates derived from Tetrahymena thermophila 60S ribosomal

subunit atomic coordinates by molecular modeling;

and

(B) a processor in electrical communication with the memory, the processor comprising a program for generating a three-dimensional model representative of the one or more target regions, and

(c) optionally further comprising a device for providing a visual representation of the model, and/or

(d) optionally further comprising a program for performing drug design.

The one or more target regions are defined by at least a part of, preferably all of the atomic coordinates in at least one of molecules 1 to 4. Preferably, the target regions are defined by less than a 1000, preferably less than 100, more preferably less than 50 or less that 30 coordinates of molecules 1 to 4.

Examples of current and preferred programs for performing drug design are MCSS (Miranker, A. and M. Karplus (1991 ) "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method." Proteins: Structure, Function and Genetics 1 1 : 29- 34, available from Molecular Simulations, Burlington, Mass) and AUTODOCK (Goodsell, D. S. and A. J. Olsen (1990) "Automated Docking of Substrates to Proteins by Simulated Annealing" Proteins: Structure, Function, and Genetics 8: 195-202, AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.)

In a preferred embodiment, the one or more target regions comprise at least a part, preferably all of an active site in the eukaryotic 60S ribosomal subunit, preferably at least a part, preferably all of a ribosomal peptidyl transferase active site or the tRNA binding sites A, P and/or E.

In a preferred embodiment of the computer system of the invention the atomic coordinates for the one or more target regions are produced by homology modeling using at least a part, preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1 A and 4A1 B for molecule 3 and (iv) 4A1 C and 4A1 D for molecule 4 of the asymmetric unit.

The term "homology modeling" refers to the practice of deriving models for three- dimensional structures of macromolecules from existing three-dimensional structures for their homologues. Homology models are obtained using computer programs that make it possible to alter the identity of residues at positions where the sequence of the molecule of interest is not the same as that of the molecule of known structure. Preferred programs used for modeling include MODELLER (Eswar et al., Comparative Protein Structure Modeling With MODELLER. Current Protocols in Bio informatics, John Wiley & Sons, Inc., Supplement 15, 5.6.1-5.6.30, 2006) and Protein Homology/analogY

Recognition Engine V 2.0 (Phyre2) (Protein structure prediction on the web: a case study using the Phyre server Kelley LA and Sternberg MJE. Nature Protocols 4, 363 - 371 , 2009).

In a further preferred embodiment, the atomic coordinates of the computer system are produced by molecular replacement using at least a part, preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1C and 4A1 D for molecule 4 of the asymmetric unit, preferably including molecular replacement of the 60S ribosomal subunit in crystals of the 80S eukaryotic ribosome, or of the 50S archaeal ribosomal subunit.

The term "molecular replacement" refers to a method for generating a model of a ribosome or ribosomal subunit whose atomic co-ordinates are unknown, preferably by orienting and positioning the atomic coordinates described herein in the unit cell of the crystals of the unknown ribosome in order to account for the observed diffraction pattern of the unknown crystal. Phases can then be calculated from this model and combined with the observed amplitudes to provide the atomic coordinates of the unknown ribosome or ribosomal subunit. This type of method is described, for example, in The Molecular Replacement Method, (Rossmann, M. G., ed.), Gordon & Breach, New York, (1972). Frequently used and preferred programs for molecular replacement include: X-PLOR (Briinger et al. (1987) Science 235:458- 460; CNS (Crystallography & NMR System, Briinger et al., (1998) Acta Cryst. Sect. D 54: 905-921 ), and AMORE: an Automatic Package for Molecular Replacement (Navaza, J. (1994) Acta Cryst. Sect. A, 50: 157- 163).

In another embodiment, the present invention relates to a computer program of the invention further comprising a program for structure determination for obtaining an electron density map of a selected ribosomal subunit, wherein

(a) the selected ribosomal subunit differs from a eukaryotic 60S ribosomal subunit comprised in a crystal of the invention, preferably the selected ribosomal subunit is the 80S eukaryotic ribosome, wherein the 60S ribosomal subunit is in complex with the 40S ribosomal subunit, or

(b) the selected ribosomal subunit differs but still is homologous to a eukaryotic 60S ribosomal subunit comprised in a crystal of the invention. Preferred selected ribosomal subunits for structure determination according to the invention by providing an electron density map are selected from the group of ribosomal subunits found in mammals, preferably human, rat, mouse, yeast, parasitic nematodes, eukaryotic multicellular pests, preferably insects, fungi, bacteria, nematodes, mites and ticks, protozoan pathogens, animal-parasitic liver flukes, etc.

Another aspect of the present invention is directed to the use of the atomic coordinates of the eukaryotic 60S ribosomal subunit of Tetrahymena thermophila and related/derived atomic coordinates for designing ligands that inhibit or modulate protein synthesis in eukaryotes.

Preferably the invention relates to the use of

a) the atomic coordinates of any one of the crystals of the invention,

(b) the atomic coordinates of the Tetrahymena thermophila 60S ribosomal subunit

deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1 C and 4A1D for molecule 4 of the asymmetric unit,

(c) the atomic coordinates of at least a part, preferably all of an active site in the

eukaryotic 60S ribosomal subunit, more preferably at least a part, most preferably all of a ribosomal peptidyl transferase active site or tRNA binding sites A, P or E,

(d) the atomic coordinates produced by molecular modeling from atomic coordinates of the Tetrahymena thermophila 60S ribosomal subunit,

(e) the atomic coordinates produced by homology modeling using at least a part,

preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1 C and 4A1 D for molecule 4 of the asymmetric unit,

(f) the atomic coordinates produced by molecular replacement using at least a part of, preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1 A and 4A1 B for molecule 3 and (iv) 4A1 C and 4A1 D for molecule 4 of the asymmetric unit, preferably including molecular replacement of the 60S ribosomal subunit in crystals of the 80S eukaryotic ribosome, or of the 50S archaeal ribosomal subunit,

(g) the atomic coordinates defined by atoms of a selected ribosomal subunit that differs from a eukaryotic 60S ribosomal subunit comprised in a crystal of the invention, preferably the selected ribosomal subunit is the 80S eukaryotic ribosome, wherein the 60S ribosomal subunit is in complex with the 40S ribosomal subunit, and/or (i) the atomic coordinates defined by atoms of a selected ribosomal subunit that differs but still is homologous to a eukaryotic 60S ribosomal subunit comprised in a crystal of the invention,

for designing ligands that inhibit or modulate protein synthesis in eukaryotes.

More preferably, the use of the invention is for designing ligands that inhibit or modulate the initiation, elongation or termination of protein synthesis in eukaryotes, preferably mammals, more preferably humans. The term "modulation" as used herein indicates any increase or decrease in as well as regulation of protein synthesis.

In a different aspect, the present invention encompasses a method for identifying a potential ligand, preferably an inhibitor or modulator of a eukaryotic 60S ribosomal subunit, preferably in complex with eukaryotic initiation factor 6 (elF6), wherein the ribosomal 60S subunit and the initiation factor are preferably from Tetrahymena thermophila, comprising the steps of:

(A) providing and using

(a) the atomic coordinates of any one of the crystals of the invention,

(b) the atomic coordinates of the Tetrahymena thermophila 60S ribosomal subunit deposited at the Protein Data Bank under accession numbers for (i) molecule 1 (4A1 E, 4A18), (ii) molecule 2 (4A17, 4A19), (iii) molecule 3 (4A1A, 4A1 B) and (iv) molecule 4 (4A1 C, 4A1 D) of the asymmetric unit,

eukaryotic 60S ribosomal subunit, preferably at least a part, preferably all of a ribosomal peptidyl transferase active site or tRNA binding sites A, P or E,

(d) the atomic coordinates derived by molecular modeling from atomic coordinates of the Tetrahymena thermophila 60S ribosomal subunit,

(e) the atomic coordinates produced by homology modeling using at least a part,

preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) for molecule 1 (4A1 E, 4A18), (ii) molecule 2 (4A17, 4A19), (3) molecule 3 (4A1 A, 4A1 B) and (iv) molecule 4 (4A1 C, 4A1 D) of the asymmetric unit,

(f) the atomic coordinates produced by molecular replacement using at least a part, preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) for molecule 1 (4A1 E, 4A18), (ii) molecule 2 (4A17, 4A19), (iii) molecule 3 (4A1A, 4A1 B) and (iv) molecule 4 (4A1 C, 4A1 D) of the asymmetric unit, preferably including molecular replacement of the 60S ribosomal subunit in crystals of the 80S eukaryotic ribosome, or of the 50S archaeal ribosomal subunit, (g) the atomic coordinates defined by atoms of a selected ribosomal subunit that differs from a eukaryotic 60S ribosomal subunit comprised in a crystal of the invention, preferably the selected ribosomal subunit is the 80S eukaryotic ribosome, wherein the 60S ribosomal subunit is in complex with the 40S ribosomal subunit, and/or

(h) the atomic coordinates defined by atoms of a selected ribosomal subunit that differs but still is homologous to a eukaryotic 60S ribosomal subunit comprised in a crystal of the invention,

for

(B) generating a three-dimensional model of at least a part of, preferably the complete eukaryotic 60S ribosomal subunit, preferably at least a part of, more preferably the complete ribosomal peptidyl transferase active site or tRNA binding sites A, P or E of the eukaryotic 60S ribosomal subunit,

(C) preferably performing whole body translations and/or rotations on the coordinates of the amino acids and/or nucleotides of the three-dimensional model of step (b),

(D) using said three-dimensional model of steps (B) and/or (C) for designing or

selecting at least one potential ligand, preferably an inhibitor or modulator of the eukaryotic 60S ribosomal subunit,

and optionally

(D) providing the at least one potential ligand,

(E) contacting the at least one potential ligand with an in vitro (no cell present) or in vivo (including living cells) test system to investigate the potential ligand ^'s ability to inhibit or modulate protein synthesis, and

(F) identifying the at least one potential ligand as a ligand or modulator, preferably an inhibitor, more preferably an antibiotic due to its ability to inhibit or modulate protein synthesis.

For step (E) it is noted that the in vivo test system is preferably an isolated cell- based or isolated tissue-based test system or a non-mammalian animal test system.

In a preferred embodiment, the method comprises as step (A) providing and using the atomic coordinates corresponding to at least part of, preferably all of the residues listed in at least one of

(i) Tables 3a, 3b and/or 3c for the A/P site of the 60S ribosomal subunit,

(ii) Tables 4a , 4b and/or 4c for the E site of the 60S ribosomal subunit, and/or

(iii) Tables 5a and/or 5b for the exit tunnel of the 60S ribosomal subunit,

preferably the atomic coordinates +2, more preferably +1.5, most preferably +1.0 A root mean square deviation (rmsd) from the backbone atoms, for generating a three- dimensional model of at least a part, preferably all of the eukaryotic 60S ribosomal subunit, preferably at least a part, preferably all of the ribosomal peptidyl transferase active site, the A/P site, the E site or tunnel exit of the ribosomal subunit of Tetrahymena thermophila, the coordinates corresponding to at least ten, preferably at least 6, more preferably at least 4 of the residues listed in at least one of tables 3a, 3b, 3c, 4a, 4b, 4c, 5a and/or 5b, and/or

Another aspect of the invention is a machine-readable medium comprising at least part of, preferably all of

(a) the atomic coordinates of any one of the crystals of the invention,

(b) the atomic coordinates of the Tetrahymena thermophila 60S ribosomal subunit

deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1C and 4A1 D for molecule 4 of the asymmetric unit,

(e) the atomic coordinates produced by homology modeling using at least a part,

preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1 C and 4A1D for molecule 4 of the asymmetric unit,

(f) the atomic coordinates produced by molecular replacement using at least a part of, preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1B for molecule 3 and (iv) 4A1 C and 4A1D for molecule 4 of the asymmetric unit, preferably including molecular replacement of the 60S ribosomal subunit in crystals of the 80S eukaryotic ribosome, or of the 50S archaeal ribosomal subunit,

(g) the atomic coordinates defined by atoms of a selected ribosomal subunit that differs from a eukaryotic 60S ribosomal subunit comprised in a crystal of the invention, preferably the selected ribosomal subunit is the 80S eukaryotic ribosome, wherein the 60S ribosomal subunit is in complex with the 40S ribosomal subunit, (h) the atomic coordinates defined by atoms of a selected ribosomal subunit that differs but still is homologous to a eukaryotic 60S ribosomal subunit comprised in a crystal of the invention, and/or

(i) the atomic coordinates corresponding to any residues listed in one or more of the tables selected from the group consisting of tables 3a, 3b, 3c, 4a, 4b, 4c, 5a and 5b and/or atomic coordinates according to any of (a) to (e) +2, preferably +1.5, more preferably +1.0 A root mean square deviation (rmsd) from the backbone atoms

and/or atomic coordinates according to any of (a) to (e) modified by performing whole body translations and/or rotations on said coordinates.

The term "residues" in the context of the invention refers to nucleotide and amino acid residues in the ribosomal subunit.

Figures

Figures 1 A to F are pictures showing the architecture of the peptidyltransferase center, the binding site of cycloheximide and the exit tunnel of the 60S subunit.

(A) Superposition of the T. thermophila 26S RNA active site region (light blue) with T. thermophilus 23S rRNA (pdb code 2WDL). Highlighted RNA elements are the active site adenosine A2808 (bacterial A2451 , red), the 8 o'clock helix, the P-loop and the T. thermophilus P-site tRNA. The N-terminus of RPL29 is displayed as a sphere.

(B) Architecture of ribosomal proteins in close vicinity to the active site.

Eukaryotic-specific extensions of RPL4, RPL 21 , RPL 0 , and RPL3 are shaded differently. The locations of the peptidyltransferase center (PTC), RPL2 and RPL29) are indicated. (Cand D) Fobs-Fcalc difference Fourier map (contoured at 3.5 σ) showing the binding site of cycloheximide at the tRNA E-site of the 60S subunit.

(C) shows a view from the inside of the subunit. The difference density reveals that the binding site of cycloheximide superimposes with A76 of the E-site tRNA, based on superposition with the archaeal 50S subunit in complex with E-site tRNA mimic. The mutations in RPL27A (L28) and RPL36A (L42) result in cycloheximide resistance. The rRNA is in grey with the proximal base C2754 (C2765 in yeast).

(D) shows a view towards the tRNA E-site (opposite direction compared to panel C). The difference density reveals that the shape and the size of the density matches the molecular structure of cycloheximide shown in the inset.

(E) Clipped view of the T. thermophila 60S ribosomal subunit showing the polypeptide exit tunnel with rRNA and proteins. RPL4 and its eukaryotic-specific extensions at the surface and in the exit tunnel (red) are shown as ribbons. Superimposed elements include an aminoacylated P-site tRNA (pdb codes 2WDL and 2WDK) and the macrolide antibiotic erythromycin (pdb code 1YI2). The position of the P site aminoacyl moiety of the tRNA is shown too.

(F) Conservation of RNA and protein elements around the exit tunnel. Eukaryotic- specific RNA and protein elements are darker, whereas conserved regions are displayed in light grey.

In the following the invention will be illustrated by means of examples, none of which are to be interpreted as limiting the scope of the invention as represented by the appended claims.

Examples

Example 1 - Cloning, expression and purification of Tetrahymenathermophila elF6

Residues 1-225 of T. thermophila elF6 were codon-optimized (GenScript USA Inc.) and cloned into an RSF1 -Duet-derived expression vector (Novagen) to produce elF6 fused to N-terminal 12x-histidine and strepll tags followed by a TEV protease cleavage site. The resulting construct was overexpressed overnight in BL21 (DE3) cells at 20°C by the addition of 1 mM IPTG. After harvesting, the cells were lysed by sonication in buffer A [20 mM Tris/HCI, pH 8.0, 500 mM KCI, 0.1 % Triton X-100 and complete EDTA-free protease inhibitors (Roche)], and cellular debris was removed by centrifugation. Soluble elF6 was applied to a cobalt-NTA sepharose column (Qiagen) pre-equilibrated in buffer A. The column was washed with buffer A supplemented with 20 mM imidazole before bound elF6 was eluted with buffer B (20 mM Tris/HCI pH 8.0, 500 mMKCI, 300mM imidazole). The N-terminal tags were removed by TEV protease digestion, leaving three amino acids of the TEV cleavage site at the N-terminus of the factor, and the buffer of the resulting sample was exchanged to buffer C (20 mM Tris/HCI pH 8.0, 500 mMKCI, 10% glycerol) and re-applied to a cobalt-NTA sepharose column (Qiagen) for removal of TEV protease, N-terminal tags and uncleaved tagged elF6.

T.thermophila elF6 was further purified by size exclusion chromatography (Superdex 75 16/60, GE Healthcare), and concentrated elF6 (7.5 mg/ml) was flash-frozen in liquid nitrogen and stored at -80°C until further use.

Example 2 - Fermentation ofT.thermophila

T. thermophila (strain 30382, American Type Culture Collection ATCC) was fermented as previously described (Rabl et al., Science 331 , 730, 2011 ). Briefly, cells were fermented in a 50 L stirred tank reactor under aerobic conditions. 0.1 g/L ampicillin was added to the growth medium(52) prior to inoculation with 1 L of pre-culture, which contained 2.4x10⁵ cells/ml. The cells were fermented at 27°C until they reached a concentration of 5.0x10⁵ cells/ml, harvested by centrifugation, washed once in

resuspension buffer RES (50 mM HEPES pH 7.6, 200 mM KCI, 10 mM MgCI₂, 5 mM EDTA, 250 mM sucrose, 2 mM DTT), and resuspended in 1.5 L of RES buffer prior to freezing in liquid nitrogen and storage at -80°C.

Example 3 - Purification of T.thermophila 60S ribosomal subunits

Frozen T.thermophila cells (120 g) were resuspended in 140 ml RES buffer supplemented with protease inhibitors. The resuspension was gently stirred in a water bath, and cellular debris was subsequently removed by centrifugation in a Sorvall SLA-1500 rotor at 4°C. The supernatant containing the 80S ribosomes was decanted and applied onto a 50% (w/w) sucrose cushion (62 mM HEPES pH 7.6, 62 mMKCI, 12 mM MgCI₂, 6 mM EDTA, 50% (w/w) sucrose, 0.025% sodium azide, 2 mM DTT), followed by centrifugation at 184.000xg and 4°C for 20 h (Beckman Ti-70 rotor).The supernatant was removed, and the pellets were resuspended in PRE buffer (50 mM HEPES pH 7.6, 10 mM KCI, 10 mM MgCI₂, 0.02% sodium azide, 2 mM DTT). The ribosomal subunits were separated on a 10%-40% (w/w) sucrose gradient (56 mM HEPES pH 7.6, 333 mM KCI, 1 1 mM MgCI₂, 2 mM DTT) by centrifugation at 103.000xg and 4°C for 14 h (Beckman SW-32 rotor). 60S ribosomal bands were harvested and pooled, the buffer was exchanged to FCB buffer (20 mM HEPES pH 7.6, 100 mM KCI, 10 mM MgCI₂, 1 mM DTT) in a centrifugal concentrator, and the sample was concentrated to 200 A₂₆₀ units/ml.

Example 4 - Complex formation

T. thermophila 60S-elF6 complexes were formed by gentle mixing of 60S ribosomal subunits (200 A₂₆₀ units/ml) with elF6 (7.5 mg/ml) at 4°C. The resulting solution contained an approximate five-fold molar excess of elF6. C12E8 (Anatrace, Maumee, USA) was added to a final concentration of 0.002% (w/v).

Example 5 - Crystallization and crvo protection

Crystallization conditions were screened using a literature-based and semi-rando- misedcrystallisation screen. Initial small crystals were observed in a condition containing 100 mM Bis-Trispropane/HCI pH 6.5, 00 mM KOAc and 6% PEG 20K at 20°C. Crystals were grown in sitting drop vapour diffusion experiments, in which 2 μΙ of ribosomal complex were mixed with 2 μΙ of reservoir solution containing 100 mM Bis-Trispropane/HCI pH 6.7, 100 mM KCI, 5.4-6.0 % PEG 14K, 3 mM spermidine. 60S-elF6 crystals grew in five days with final dimensions up to 600 pm x 400 pm x 80 pm. Addition of 1 mM of the antibiotic cycloheximide during crystallization resulted in morphologically improved crystals of larger size, which displayed better diffraction properties (see below). Dehydration and cryoprotection of crystals was achieved either in the presence (60S-elF6- cycloheximide complex) or absence (60S-elF6 complex) of 1 mM cycloheximide by stepwise addition of PEG 14K and ethylene glycol as cryoprotectant to final

concentrations of 15% PEG 14K and 25% ethylene glycol. Crystals were mounted and plunged into liquid nitrogen.

Example 6 - Data collection and processing

X-ray diffraction data was collected at the beamline X06SA of the Swiss Light Source (SLS) at the Paul Scherrer Institut (PSI) in Villigen using the PILATUS 6M detector. Datasets were integrated and scaled using the XDS package ( W. Kabsch, Xds. Acta Crystallogr D Biol Crystallogr66, 125, 2010). The initial 60S-elF6 crystals, which diffracted to 3.7 A resolution, belong to space groups P2i with unit cell dimension of a=320.5A, 6=288.9 A, c=535.7A and with a β angle of 91.506°. Comparison with 3.5 A data obtained from a crystal containing the 60S-elF6-cycloheximide complex (P2i, a=320.2 A, 6=289.3 A, c=535.0A, β=91.220) showed that the two datasets were isomorphous, with no obvious differences in the overall structure or the region around the bound antibiotic. Therefore, the final model was refined against the 3.5 A 60S-elF6- cycloheximide dataset (see Table S1 below).

Example 7 - Structure determination, model building and refinement

Initial molecular replacement solutions were obtained using PHASER (McCoy et al., Phaser crystallographic software. J Appl Crystallogr 40, 658, 2007) and the Haloar- culamaris mortui 50S structure (pdb code 1jj2) as a search model, which revealed four copies per asymmetric unit (ASU) in the crystal (molecules 1-4). The molecules are arranged as two pairs, which pack against each other via the small subunit binding face. The archaeal model was subdivided into -200 rigid body and TLS groups per subunit and refined in PHENIX using non-crystallographic (NCS) symmetry restraints for residue- wise B-factor and individual coordinate refinement (Adams et al., PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 58, 1948, 2002). The appearance of the resulting F_0bS-F_cai_c difference Fourier density map of eukaryotic-specific additional RNA and protein parts was improved by subjecting the phases to 4-fold NCS averaging and solvent flipping in CNS (Brunger et al., Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54, 905, 1998; A. T. Brunger, Version 1.2 of the Crystallography and NMR system. Nat Protoc 2, 2728, 2007). The initial averaging mask was iteratively updated by positioning the protein structures of homoiogs solved in isolation and RNA expansion segments from the yeast 80S EM models

(Armache et al., Proc. Natl. Acad. Sci. U.S.A. 107, 19748, 2010; Armache et al., Proc. Natl. Acad. Sci. U.S.A. 107, 19754, 2010) or a recent X-ray structure (Ben-Shem et al., Science 330, 1203, 2010) together with manual model building of novel parts using COOT and O (Emsley et al., Acta Crystallogr D Biol Crystallogr 66, 486, 20 0; Jones et al. Acta Crystallogr A47 (Pt 2), 110, 1991 ). The resulting electron density maps allowed re-tracing and building of a full atomic model. For most of the model, molecule 2 was used for building, aided by inspection of density in molecules 1 , 3 and 4. For correct sequence assignment of the proteins, positions of bulky side chains, predicted secondary structure patterns (Kelle and Sternberg, Nat Protoc 4, 363, 2009) and locations of zinc ions were used as landmarks. The precise zinc positions were determined by calculating an anomalous difference Fourier map. In most regions of the 60S subunit, bulky and ordered side chains were clearly visible at 3.5 A, whereas in some less well ordered parts on the solvent exposed surface, the precise side chain conformations could not be determined. It was possible to build the RNA with confidence at 3.5 A resolution, since the phosphate backbone was clearly visible and stacked bases were usually separated, allowing differentiation between purines and pyrimidines in most regions of the structure. Hexa-coordinated magnesium ions were positioned into strong isolated Fourier difference density peaks.

At later stages, NCS averaging was no longer required, and the model was completed by several rounds of rebuilding into Fourier difference maps followed by refinement using PHENIX (see Adams et al. above) and CNS (see Brunger et al. and A. T. Brunger above). The final model was refined to working and free R-factors of 2 .6 % and 24.5 %, with excellent geometry (see Table S1 ), using PHENIX (see Adams et al. above). The refinement strategy included anisotropic scaling, bulk solvent correction, group wise NCS restrained individual coordinate and residue-wise B factor refinement (46 NCS groups comprising individual rRNAs or proteins), and anisotropic TLS B factor refinement for the RNA and protein parts (1472 automatically determined TLS groups). For the grouped B factor refinement of the hexa-coordinated magnesiums, each

Mg²⁺*6H₂0 was treated as one group. Automatically determined protein Ramachandran and secondary structure restraints and automatically generated and manually edited RNA base-pair restraints (including the entire Saenger classification) were applied throughout. The final model includes the entire 26S rRNA except for nucleotides 1258-1305 (stalk), 1786-1791 (ES24 loop), 1975-2096 (ES27), 2441 -2497(L1 protuberance) and 3353-3354 (the last two terminal bases), and it comprises residues 1 -120 from the 5S rRNA and 1-154 of the 5.8S rRNA. It further contains most proteins with archaeal or bacterial homologues and additional eleven proteins (RPL13, RPL18A (L20), RPL22, RPL27, RPL28, RPL29, RPL35A (L33), RPL34, RPL36, RPL38, RPL40), which are not present in previous crystal structures of archaeal, bacterial or eukaryotic ribosomes (see Table S2) (Ban et al., Science 289, 905, 2000; Selmer et al., Science 313, 935, 2006; Ben-Shem et al., Science 330, 1203, 2010). Proteins that were not built are acidic proteins RPLP1 , RPLP2, the small peptide RPL41 , and proteins associated with flexible RNA parts [RPL10A (L1 ) and RPL12 (L12)]. A predicted insertion in T .thermophila

RPLPO compared to the Thermotoga maritime homolog (pdbcodelzax) is omitted, as the protein is not ordered well enough for rebuilding. Therefore, RPLPO (RPP0) was modelled and refined as poly-serine of 1 zax. The C terminal a helix of protein RPL19 is present in different conformations and visible to different extents in the four molecules in the ASU due to crystal contacts. The amino acid sequence of the terminus could be assigned in one of the molecules, whereas it was refined as poly-serine in two other copies (Table S2).

Closer inspection of the crystal contacts between the four 60S subunits in the ASU arranged in a pair-wise manner revealed that each pair is coordinated by two molecules of elF6, which in turn interact with two further elF6 molecules from the second 60S pair.

Example 8 - Calculation of buried surface areas and surface potentials.

Buried surface areas between ribosomal proteins were calculated using PISA (Krissinel and Henrick, J Mol Biol 372, 774, 2007). The surface potential of RPL22 was calculated and visualized with PYMOL (W. L. DeLano, 2002, www.pymol.org).

Example 9 - Structure superpositions and sequence alignments

Structural superpositions were performed using PYMOL (W. L. DeLano, 2002, www.pymol.org) and O (Jones et al., Acta Crystallogr A47 (Pt 2), 1 10, 1991 ). Figures were generated (i) using O with an LSQ alignment of H. marismortui 23S rRNA2089- 2109 (pdb code 1 yi2) and Ti thermophila 26S rRNA 2385-2405 resulting in an rmsd of 0.988A and (ii) using O with an LSQ alignment of T. thermophilics 23S rRNA (pdb code 2wdl) including residues 2441 -2461 and T. thermophila 26S rRNA residues 2798-2818, resulting in an rmsd of 0.73A. The Esite tRNA mini helix (pdbcode 1qvf) was positioned according to an LSQ alignment of H. marismortui 23S rRNA residues 2423-2463

(pdbcode 1qvf) with T. thermophila 26S rRNA residues 2745-2785 in O, resulting in an rmsd of 1.25θΑ. Sequences were aligned using BLAST (Altschul et al., Basic local alignment search tool. J Mol Biol215, 403 (1990) and CLUSTALW (Larkin et al., Clustal W and Clustal X version 2.0. Bioinformatics23, 2947 (2007)) and visualized with Jalview (www.jalview.org).

Example 10 - Sequencing of T. thermophila 26S and 5.8S rRNA

While building the atomic model of the 26S rRNA, considerable discrepancies between the deposited DNA sequence (GenBank: X54512.1 ) and the observed electron density became apparent. To verify the rDNA sequences of the 26S and 5.8S rRNAs, the identical extrachromosomal copies of rDNA were isolated from T. thermophila cells using a Plasmid Maxi Prep (Qiagen). The region containing the 26S and 5.8S rDNA was amplified using Phusion DNA polymerase (Finnzymes) supplemented with dUTPase from Pyrococcusabyssii, and the resulting PCR product was directly used for sequencing (Microsynth AG, Switzerland). The DNA sequence obtained by sequencing shows that the previous entry (GenBank: X54512.1 ) contains numerous base substitutions as well as insertions and deletions. The new sequence is a closer match to the related

Tetrahymena species Tetrahymenapyriformis (GenBank: X54004.1 ) and is consistent with base pairing in the secondary structure and the observed electron density. The new sequence has been deposited with the GenBank accession code JN547815.

Example 1 1 - Sequence annotation

For all T. thermophila 60S ribosomal proteins, correctly spliced open reading frames could be detected by using tbiastn of the BLAST interface (blast.ncbi.nlm.nih.gov) and searching the deposited ESTs of T. thermophila either with already annotated T. thermophila GenBank entries or homologous humanor Paramecium tetraurelia

sequences. The correct length of the assignments could be further verified by multiple sequence alignments of eukaryotic ribosomal proteins using BLAST and by direct inspection of the electron density maps during protein building. The human protein names assigned by the UNIPROT database were used (see Table S2). For easy and direct comparison with homologous yeast and archaeal ribosomal proteins, the corresponding yeast names are indicated in parantheses throughout the text where they differ in name, and both the yeast and archaeal homologs are listed in Table S2.

Example 12 - Figure generation Figures for showing atomic models can be generated using O (Jones et al., Acta Crystallogr A47 (Pt 2), 1 10, 1991 ).) and PYMOL (W. L. DeLano, 2002, www.pymol.org).

Example 13 - Structure representation

A PYMOL script that displays the two PDB files corresponding to one complete 60S subunit and labels the proteins according to the UNIPROT, yeast and E. coli nomenclatures is available on Science Online. Other scripts, movies and related material have been made available on http://www.mol.biol.ethz.ch/groups/ban_group/Ribosome.

Tables

Table 1. Data collection and model statistics.

Crystal form P2i (60S-elF6-cycloheximide)

Unit cell dimensions (A³) 320.2 x 289.3 x 535.0

α = γ = 90°, β = 91.220°

Molecules / ASU 4

Solvent content (%) ^a 55

Data collection

Wavelength (A) 1.00

Temperature (K) 100

Resolution (A) ^b 40 - 3.52 (3.72 - 3.52)

Unique reflections⁰ 1 192534

Redundancy 3.7

Rmerge (%) 13.4 (80.9)

Completeness (%) ^b 99.3 (99.6)

l / a ^b 8.8 (1.6)

Model statistics

Model composition:

nonhydrogenatoms 51 1402

proteinresidues 27032

RNA bases 13572

ligands (Zn²⁺ / Mg²⁺'6H₂0) 20/ 865

Refinement:

resolution (A) 20.0 - 3.52

total number of reflections 1 186656

test reflections (%) 9951 (0.84)

Rcryst Rfree (%) ^ 21.6 / 24.5

average B value (A²) 108.2

Rmsdeviations:

bonds (A) 0.009

angles (°) 1.14

dihedrals (°) 19.39

Ramachandran plot (70):

favored (%) 22457 (93.5)

allowed (%) 1531 (6.4)

generously allowed (%) 24 (0.1 )

disallowed (%) 4 (0.0)

l(hj) - [l(h)]| /∑l(h,i), where [1(h)] is the mean intensity of the reflections. aEstimated solvent content used during averaging

"Values for highest resolution shells are given in parentheses.

⁰ For nativelyscaled data, Bijvoet pairs were merged.

dRcryst and Rf_ree were calculated from the working and test reflection sets. Table 2: Protein segments included in the atomic model together with accession numbers and related homologs.

Tetrahym UNIPROT E. co//

chain residues in length MW yeast

enatherm accession conservation'"' name archaeal homolog PDB related structures PDB fold

ID model (aa) (Da)

ophila^w code «0 name'⁰'

poly-

RPL 1zax:

0 G 4-126 serin e of EAB RPL10 RPP0 T. maritime L10

P A

Izax"¹

RPL 3 B 2-387 096774 391 44248 EAB RPL3 RPL3 H. marismortui L3 1jj2:B

RPL 4 C 2-410 P0DJ55 410 45107 EAB RPL4 RPL4 H. marismortui L4 1jj2:C

1jj2:

RPL 5 M 2-301 Q231U7 301 34454 EAB RPL18 RPL5 H. marismortui L18p

M

S. solfataricus

RPL 6 e 2-191 P0DJ56 191 21732 E RPL6 n/a SH3-like ba

L14e

RPL 7 V 6-239 P0DJ13 239 27614 EAB RPL30 RPL7 H. marismortui L30 1j]2:V

H. marismortui

RPL 7A F 19-249 P0DJ14 255 29069 EA RPL8 1jj2:F

L7AE

RPL 8 A 2-258 P0DJ52 264 28650 EAB RPL2 RPL2 H. marismortui L2 1jj2:A

RPL 9 E 1-186 Q22AX5 188 21336 EAB RPL6 RPL9 H. marismortui L6 1jj2:E

RPL 10 H 2-101 , 113-213 Q235 8 215 24459 EAB RPL16 RPL10 H. marismortui L10e 1jj2:H

RPL 11 D 4-172 P24119 172 19718 EAB RPL5 RPL1 1 H. marismortui L5 1jj2:D

gyrase (rmsd 3.7A) 3cwv:

RPL 13 u 1-203 P0DJ58 206 23337 EA RPL13 2-layer sand

A

13

RPL I 1-198 P0DJ15 198 22593 EAB RPL13 RPL16 H. marismortui L13 1J|2:I

A

2joy:

RPL 14 f 2-126 Q24C27 126 14598 EA RPL14 S. solfataricus L14e

A

RPL 15 L 2-204 Q22A30 204 24076 EA RPL15 H. marismortui L15e 1jj2:L

1jj2:

RPL 17 Q 2-158 P0DJ16 183 20375 EAB RPL22 RPL 7 H. marismortui L22

Q

RPL 18 N 2-181 P0DJ17 181 20589 EA RPL18 H. marismortu^&e 1jj2:N

M.

18

RPL X 2-189 P0DJ18 189 21978 EA RPL20 thermoautotrophicu 2jxt:A

A

m Lx

2-185/176/

RPL 19 0 P0DJ60 185 21373 EA RPL19 H. marismortui L19 1jj2:

147/154 ^<0 0

RPL 21 P 2-157 Q23TC8 157 18123 EA RPL21 H. marismortui L21e 1jj2:P

CopA ATPase

RPL 22 m 12-111 Q23BV5 118 13780 E RPL22 n/a 1oq3:A 2-layer sand

(rmsd 2.2A) ^(d)

RPL 23 J 4-141 P0DJ53 141 14880 EAB RPL14 RPL23 H. marismortui L14p 1p:J

23

RPL R 30-150 P0DJ57 150 16862 EAB RPL23 RPL25 H. marismortui L23 1jj2:R

A

(a) nomenclature according to the ribosomal protein gene database (22).

(b) abbreviations: E, present in eukaryotes; EA, present in eukaryotes and archaea; EAB, present in eukaryotes, archaea and bacteria

(c) construct contains residues 1-225, and 3 residues of the.purification tag remain at the N-terminus after TEV cleavage

(d) found by using PDBeFold (Krissinel & Hendrick, Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions, Acta Christallogr D Biol Crystallogr 60, 22 2004).

(e) found by using the DALI server (Holm and Rosenstrom, Dali server: conservation mapping in 3D. Nucleic Acids Res, 38, W545, 2010)).

(f) protein RPLP and the C-termini of RPL19 in subunits 2 and 4 were refined as poly-serine and deposited as UNK residues

Table 2 (continued): Protein segments included in the atomic model together with accession numbers and related homologs.

A)

u 0 A) ^m

c

U

A) <« (aPa)

(a) Nomenclature according to the ribosomal protein gene database (Nakao et al.. Nucleic Acids Res 32, D 168 (2004).

(c) construct contains residues 1 -225, and 3 residues of the purification tag remain at the N-terminus after TEV cleavage

(d) found by using PDBeFold (Krissinel and Hendrick, Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions, Acta Christallogr D Biol

Crystallogr 60, 2256, 2004)

(c) found by using the DALI server (Holm and Rosenstrom, Dali server: conservation mapping in 3D. Nucleic Acids Res, 38, W545, 2010).

The following tables 3 to 5 list the residues within the vicinity of the A/P site (Tables 3a-c), the E site (Tables 4a-c) and the exit tunnel (Tables 5a & b). Residues in the vicinity of these sites were selected as follows:

The pdb coordinates 1fg0, which contain a puromycin ligand, were superimposed onto the Tetrahymena thermophila 60S subunit (pdb coordinates 4a17 and 4a19).

Residues surrounding the position of puromycin in the context of the 60S subunit were selected with increasing distances (0-5, 5-10 and 10-15 Angstroms). Similarly, an E-site mini helix from pdb coordinates 1qvf was superimposed onto the Tetrahymena thermophila 60S subunit (pdb coordinates 4a17 and 4a19).

The position of the terminal base of the mini helix (A76) was used as a reference to define the E-site. Residues surrounding A76 in the context of the 60S subunit were selected with increasing distances (0-5, 5-10 and 10-15 Angstroms). The position of the exit tunnel was defined by superimposing a model of the TNAC leader sequence

(contained within pdb coordinates 2wqq) onto the Tetrahymena thermophila 60S subunit (pdb coordinates 4a17 and 4a19). The TNAC leader sequence was used as a reference, from which surrounding residues were selected (0-5 and 5-10 Angstroms)."

For understanding the following tables it is noted that the nucleotide residues refer to the four bases of the Tetrahymena thermophila 26S rRNA with their numbering according to the sequence presented in the published sequence, i.e. genbank entry JN547815.1 , genbank sequence link: http://www.ncbi.nlm.nih.gOv/nuccore/JN547815.1.

The residues in the vicinity of the A/P site

Table 3a Table 3b Table 3c

0-5 Angstroms 5-10 Angstroms 10-15 Angstroms

G2398 C2282 C1928

C2400 A2399 C2279

A2807 C2401 C2280

A2808 G2803 U2281

C2809 G2804 C2282

U2863 U2810 U2293

C2864 U2849 A2392

G2910 U2850 A2393

C2930 G2851 G2395

G2940 U2857 A2396

U2941 C2858 A2397

U2942 A2860 G2398

U2861 A2399

G2862 C2402

G2865 U2403 U2911 G2608

U2912 G2609

C2930 A2796

G2933 A2799

G2939 C2800

A2959 A2801

G2960 G2802

G2961 U2806

U2962 G2811

G2812

U2848

G2851

A2852

U2853

C2854

C2855

U2856

G2859

G2866

U2909

C2913

A2914

G2927

A2928

A2929

G2931

U2932

C2936

U2937

G2938

G2940

U2943

C2958

U2963

G2965

U2967

RPL3 residue W253 (eab)*

*Tryptophan 253 of the ribosomal protein RPL3 is within 15 Angstroms of the A/P site. This protein is present in eukaryotes (e), archaea (a) and bacteria (b), hence (eab).

The residues in the vicinity of the E site

**The E-site has different proteins in its vicinity. Phenylalanine 56 (F56) of RPL36A, which is called RPL42 in yeast, is within 5 Angstroms of the E site. This protein is only present in eukaryotes and archaea, hence the comment ea. With increasing distance, more and more residues of RPL36 are selected and RPL36A was used as a subheading for all residues listed subsequently. Table 4a Table 4b Table 4c

0-5 Angstroms 5-10 Angstroms 10-15 Angstroms

C2753 G2750 G39

C2754 A2751 A41

G2781 U2752 G89

G2782 U2755 G90

RPL36A_F56 (yeast name: RPL42), ea U2756 C91

G2779 G92

A2780 C984

U2783 C2415

G2788 U2416

A2789 C2417

A2790 G2640

RPL36A (yeast name RPL42), ea

residues: U2641

K28 G2720

E31 G2721

R38 C2749

Y41 C2754

Q51 U2757

K53 A2778

P54 G2784

155 C2786

R57 A2787

K58 A2791

K59 A2792

RPL36A (yeast name RPL42), ea

A60 residues:

K61 Q25

T63 Y26

K64 K27

K65 S29

R85 K30

S32

T33

A35

Q36

G37

R39

R40

D42

K44

Q45

T52 T62

V66

A67

L68

V80

181

P82

183

K84

C86

RPL27A(yeast L28, Ecoli L15) - eab residues: ***

M40

H41

R44

G56

K57

PL18 (yeast L18), ea****

H178

G179

L180

K181

As the radius around the E site increases, two more proteins are close to the E site.

***RPL27A, which is called L28 in yeast and LI 5 in E. coli, is present in all domains of life, hence the designation eab. Five of its residues are within 15 Angstroms of the E site (M40, H41 , R44, G56 and K.57). ****RPL18, which is only present in eukaryotes and archaea (hence ea), and which is also called LI 8 in yeast, has four residues, which are positioned within 15 Angstroms of the active site.

The exit tunnel amino acids

Table 5a Table 5b

0-5 Angstroms 5-10 Angstroms

C1870 A354

G2395 C355

A2396 U356

G2398 A364

A2399 A365

C2400 A366

A2808 G903

G2862 U904

U2863 U905

U2942 A908

U2966 A909

RPL39 (yeast 139), ea, residues:5* A946

Y37 U1465

RPL4_eab, residues:6* G1466 W72 G1467

R76 A1529

T89 A1530

H90 C1531

S92 A2135

693 U2136

RPL17(yeast L17, Ecoli L22),eab,

residues 7* C2353

R130 C2354

A2394

A2397

C2401

A2796

C2798

A2799

G2804

A2807

C2809

C2858

A2860

U2861

C2864

G2933

U2941

U2943

U2967

U2968

U2969

RPL39 (yeast L39), ea, resid

R36

N38

S39

K40

R41

R46

T47

RPL4_eab, residues:

S71

G73

T74

G75

A77

V78

R83 V84

G85

G86

S87

G88

91

Q94

A95

RPL17(yeast L17, Ecoli L22),eab,

residues

Q125

R128

R129

Y132

A134

H135

G136

R137

1138

N139

P140

In the vicinity of the exit tunnel, there are numerous proteins, which include:

5* RPL39, which is present in eukaryotes and archaea (hence ea) and also called L39 in yeast.

6* RPL4, which is present in all domains of life (hence eab) with the same name in yeast etc.

7* RPL17, which is present in all domains of life (hence eab), which is also called L17 in yeast but L22 in E coli

Claims

1. A crystal of the eukaryotic 60S ribosomal subunit of Tetrahymena thermophila in complex with the eukaryotic initiation factor 6 (elF6) of Tetrahymena thermophila, comprising an average thickness greater than 10 μιτι and diffracting X-rays to a resolution of at least 7.0, preferably 3.5 A.

2. The crystal of claim 1 having a monoclinic space group P2^ with unit cell

dimensions of a=320.5±10 A, 6=289±10 A, c=536±10 A and with a β angle of 91.5±0.5 °.

3. The crystal of claim 1 or 2, preferably claim 1 , wherein the crystal structure is

defined by the coordinates deposited under Protein Data Bank accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1 C and 4A1 D for molecule 4 of the asymmetric unit.

4. The crystal of any of claims 1 to 3, preferably claim 1 , consisting of the eukaryotic 60S ribosomal subunit in complex with eukaryotic initiation factor 6 comprising a ligand, preferably in complex with the eukaryotic 60S ribosomal subunit and/or the eukaryotic initiation factor as defined by the coordinates deposited under Protein Data Bank accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1 A and 4A1 B for molecule 3 and (iv) 4A1C and 4A1 D for molecule 4 of the asymmetric unit.

5. The crystal of claim 4, wherein the ligand is an inhibitor or modulator of protein

synthesis, preferably an antibiotic, more preferably selected from the group consisting of chloramphenicols, macrolides, lincosamides, streptogramins, althiomycins, oxazolidinones, nucleotide analogs, thiostreptons (including the micrococcin family), peptide antibiotics, glutarimides, trichothecenes, TAN -I OS7, pleuromutilins, hygromycins, betacins, eveminomicins, boxazomycins and fusidanes, most preferably selected from the group consisting of puromycin, cycloheximide and chloramphenicol.

6. The crystal of any of claims 1 to 5, preferably any of claims 1 , 2 and 5, wherein the average thickness of the crystal is 10 to 600 pm, preferably an average thickness selected from the group consisting of 10 to 50, 51 to 100, 101 to 150, 151 to 200, 100 to 300 and 300 to 600 pm.

7 The crystal of any of claims 1 to 8, preferably any of claims 1 , 2 and 5, wherein the eukaryotic 60S ribosomal subunit is homologous to the eukaryotic 60S ribosomal subunit from Tetrahymena thermophila, and the similarity, preferably identity is at least 30, more preferably at least 70 or 80, most preferably 90, 95 or 99 % identity to the Tetrahymena thermophila ribosomal RNA and ribosomal proteins that they have in common.

8. A method of producing a crystal according to any of claims 1 to 7, comprising the steps of:

(d) 50 to 200, preferably 75 to 150, more preferably about 100 mM Bis-Tris

propane, preferably Bis-Tris propane HCI,

(e) 50 to 200, preferably 75 to 150, more preferably about 100 mM acetate,

preferably potassium acetate,

(f) 3 to 10, preferably 5 to 7, more preferably about 6 % by weight PEG

(polyethylene glycol), preferably PEG 20K

at a temperature of 15 to 30, preferably 18 to 25, more preferably about 20 °C, and a pH of 5.5 to 7.5, preferably 6 to 7, more preferably about 6.5.

9. The method of claim 8, comprising further growth crystallization comprising the steps:

(iii) mixing about equal aqueous amounts of the 60S ribosomal subunit elF6

complex with

(aii) 50 to 200, preferably 75 to 150, more preferably about 100 mM KCI, (aiii) 4.5 to 7, preferably 5.4 to 6.0 % by weight PEG, preferably PEG 14K, (aiv) 1 to 5, preferably 2 to 4, more preferably about 3 mM spermidine, preferably by the sitting drop vapor diffusion method and at a pH of 6 to 7.5, preferably 6.5 to 7, more preferably about 6.7, and optionally adding 0.5 to 2, preferably about 1 mM of a ribosomal ligand, preferably an antibiotic, preferably cycloheximide during growth crystallization.

10. A composition comprising a crystal according to any of claims 1 to 7 and/or a

crystal produced according to claims 8 or 9.

11. A computer system comprising:

(A) a memory storing data defining one or more target regions

(a1 ) by atomic coordinates of the Tetrahymena thermophila 60S ribosomal subunit deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A 7 and 4A19 for molecule 2, (iii) 4A1 A and 4A1 B for molecule 3 and (iv) 4A1 C and 4A1 D for molecule 4 of the asymmetric unit, and/or

(a2) by atomic coordinates derived from Tetrahymena thermophila 60S

ribosomal subunit atomic coordinates by molecular modeling; and

(d) optionally further comprising a program for performing drug design.

12. The computer system of claim 11 , wherein the one or more target regions comprise at least a part, preferably all of an active site in the eukaryotic 60S ribosomal subunit, preferably at least a part, preferably all of a ribosomal peptidyl transferase active site or the tRNA binding sites A, P and/or E.

13. The computer system of claim 1 1 or 12, wherein the atomic coordinates are

produced by homology modeling using at least a part, preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1 C and 4A1D for molecule 4 of the asymmetric unit.

14. The computer system of any of claims 1 1 to 13, preferably claim 1 1 , wherein the atomic coordinates are produced by molecular replacement using at least a part, preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1 C and 4A1D for molecule 4 of the asymmetric unit, preferably including molecular replacement of the 60S ribosomal subunit in crystals of the 80S eukaryotic ribosome, or of the 50S archaeal ribosomal subunit.

15. The computer system of any of claims 1 1 to 14, preferably claim 11 , further

comprising a program for structure determination for obtaining an electron density map of a selected ribosomal subunit, wherein

(a) the selected ribosomal subunit differs from a eukaryotic 60S ribosomal subunit comprised in a crystal of any of claims 1 to 4, preferably the selected ribosomal subunit is the 80S eukaryotic ribosome, wherein the 60S ribosomal subunit is in complex with the 40S ribosomal subunit, or

(b) the selected ribosomal subunit differs but still is homologous to a eukaryotic 60S ribosomal subunit comprised in a crystal of any of claims 1 to 4.

16. Use of

a) the atomic coordinates of any one of the crystals according to one of claims 1 to 7, preferably claim 3,

(b) the atomic coordinates of the Tetrahymena thermophila 60S ribosomal subunit deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1C and 4A1 D for molecule 4 of the asymmetric unit,

(c) the atomic coordinates of at least a part, preferably all of an active site in the eukaryotic 60S ribosomal subunit, more preferably at least a part, most preferably all of a ribosomal peptidyl transferase active site or tRNA binding sites A, P or E,

(d) the atomic coordinates produced by molecular modeling from atomic

coordinates of the Tetrahymena thermophila 60S ribosomal subunit,

(e) the atomic coordinates produced by homology modeling using at least a part, preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1C and 4A1 D for molecule 4 of the asymmetric unit,

(g) the atomic coordinates defined by atoms of a selected ribosomal subunit that differs from a eukaryotic 60S ribosomal subunit comprised in a crystal of any of claims 1 to 4, preferably the selected ribosomal subunit is the 80S eukaryotic ribosome, wherein the 60S ribosomal subunit is in complex with the 40S ribosomal subunit, and/or

(i) the atomic coordinates defined by atoms of a selected ribosomal subunit that differs but still is homologous to a eukaryotic 60S ribosomal subunit comprised in a crystal of any of claims 1 to 4,

for designing ligands that inhibit or modulate protein synthesis in eukaryotes.

17. Use according to claim 16 for designing ligands that inhibit or modulate the

initiation, elongation or termination of protein synthesis in eukaryotes, preferably mammals, more preferably humans.

18. A method for identifying a potential ligand, preferably an inhibitor or modulator of a eukaryotic 60S ribosomal subunit, preferably in complex with eukaryotic initiation factor 6 (elF6), wherein the ribosomal 60S subunit and the initiation factor are from Tetrahymena thermophila, comprising the steps of:

(A) providing and using

(a) the atomic coordinates of any one of the crystals according to one of claims 1 to 7, preferably claim 3,

(c) the atomic coordinates of at least a part, preferably all of an active site in the eukaryotic 60S ribosomal subunit, preferably at least a part, preferably all of a ribosomal peptidyl transferase active site or tRNA binding sites A, P or E,

(d) the atomic coordinates derived by molecular modeling from atomic

coordinates of the Tetrahymena thermophila 60S ribosomal subunit,

(e) the atomic coordinates produced by homology modeling using at least a part, preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) for molecule 1 (4A1 E, 4A18), (ii) molecule 2 (4A17, 4A19), (3) molecule 3 (4A1 A, 4A1 B) and (iv) molecule 4 (4A1 C, 4A1 D) of the asymmetric unit,

(f) the atomic coordinates produced by molecular replacement using at least a part, preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) for molecule 1 (4A1 E, 4A18), (ii) molecule 2 (4A17, 4A19), (iii) molecule 3 (4A1A, 4A1 B) and (iv) molecule 4 (4A1 C, 4A1 D) of the asymmetric unit, preferably including molecular replacement of the 60S ribosomal subunit in crystals of the 80S eukaryotic ribosome, or of the 50S archaeal ribosomal subunit,

(h) the atomic coordinates defined by atoms of a selected ribosomal subunit that differs but still is homologous to a eukaryotic 60S ribosomal subunit comprised in a crystal of any of claims 1 to 4,

for

(E) generating a three-dimensional model of at least a part of, preferably the

complete eukaryotic 60S ribosomal subunit, preferably at least a part of, more preferably the complete ribosomal peptidyl transferase active site or tRNA binding sites A, P or E of the eukaryotic 60S ribosomal subunit,

(F) preferably performing whole body translations and/or rotations on the

coordinates of the amino acids and/or nucleotides of the three-dimensional model of step (b),

(G) using said three-dimensional model of steps (B) and/or (C) for designing or selecting at least one potential ligand, preferably an inhibitor or modulator of the eukaryotic 60S ribosomal subunit, and optionally

(D) providing the at least one potential ligand, (E) contacting the at least one potential ligand with an in vitro or in vivo test system to investigate the potential ligand ^'s ability to inhibit or modulate protein synthesis, and

19. A method according to claim 18, comprising as step

(A) providing and using the atomic coordinates corresponding to at least part of, preferably all of the residues listed in at least one of

(i) Tables 3a, 3b and/or 3c for the A/P site of the 60S ribosomal subunit,

(iii) Tables 5a and/or 5b for the exit tunnel of the 60S ribosomal subunit, preferably the atomic coordinates +2, more preferably +1.5, most preferably +1.0 A root mean square deviation (rmsd) from the backbone atoms,

for generating a three-dimensional model of at least a part, preferably all of the eukaryotic 60S ribosomal subunit, preferably at least a part, preferably all of the ribosomal peptidyl transferase active site, the A/P site, the E site or tunnel exit of the ribosomal subunit of Tetrahymena thermophila, the coordinates corresponding to at least ten, preferably at least 6, more preferably at least 4 of the residues listed in at least one of tables 3a, 3b, 3c, 4a, 4b, 4c, 5a and/or 5b.

20. Machine-readable medium comprising at least part of, preferably all of

(a) the atomic coordinates of any one of the crystals of the invention,

(b) the atomic coordinates of the Tetrahymena thermophila 60S ribosomal subunit deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1 A and 4A1 B for molecule 3 and (iv) 4A1C and 4A1 D for molecule 4 of the asymmetric unit,

(d) the atomic coordinates produced by molecular modeling from atomic

coordinates of the Tetrahymena thermophila 60S ribosomal subunit, (e) the atomic coordinates produced by homology modeling using at least a part, preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1C and 4A1D for molecule 4 of the asymmetric unit,

(f) the atomic coordinates produced by molecular replacement using at least a part of, preferably all of the atomic coordinates deposited at the Protein Data Bank under accession numbers (i) 4A1 E and 4A18 for molecule 1 , (ii) 4A17 and 4A19 for molecule 2, (iii) 4A1A and 4A1 B for molecule 3 and (iv) 4A1C and 4A1 D for molecule 4 of the asymmetric unit, preferably including molecular replacement of the 60S ribosomal subunit in crystals of the 80S eukaryotic ribosome, or of the 50S archaeal ribosomal subunit,

(g) the atomic coordinates defined by atoms of a selected ribosomal subunit that differs from a eukaryotic 60S ribosomal subunit comprised in a crystal of any of claims 1 to 4, preferably the selected ribosomal subunit is the 80S

eukaryotic ribosome, wherein the 60S ribosomal subunit is in complex with the 40S ribosomal subunit,

(h) the atomic coordinates defined by atoms of a selected ribosomal subunit that differs but still is homologous to a eukaryotic 60S ribosomal subunit comprised in a crystal of any of claims 1 to 4, and/or

(i) the atomic coordinates corresponding to any residues listed in one or more of the tables selected from the group consisting of tables 3a, 3b, 3c, 4a, 4b, 4c, 5a and 5b

and/or atomic coordinates according to any of (a) to (e) +2, preferably +1.5, more preferably +1.0 A root mean square deviation (rmsd) from the backbone atoms and/or atomic coordinates according to any of (a) to (e) modified by performing whole body translations and/or rotations on said coordinates.