CN113912668B - In-situ crystallization device and method - Google Patents
In-situ crystallization device and method Download PDFInfo
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- CN113912668B CN113912668B CN202111259671.6A CN202111259671A CN113912668B CN 113912668 B CN113912668 B CN 113912668B CN 202111259671 A CN202111259671 A CN 202111259671A CN 113912668 B CN113912668 B CN 113912668B
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- 238000011065 in-situ storage Methods 0.000 title claims abstract description 326
- 238000002425 crystallisation Methods 0.000 title claims abstract description 149
- 230000008025 crystallization Effects 0.000 title claims abstract description 143
- 238000000034 method Methods 0.000 title claims abstract description 42
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 239000011248 coating agent Substances 0.000 claims description 31
- 238000000576 coating method Methods 0.000 claims description 31
- 239000013078 crystal Substances 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 22
- 238000007789 sealing Methods 0.000 claims description 17
- 238000012835 hanging drop method Methods 0.000 claims description 12
- 230000000295 complement effect Effects 0.000 claims description 9
- 239000002390 adhesive tape Substances 0.000 claims description 8
- 239000012452 mother liquor Substances 0.000 claims description 8
- 239000011159 matrix material Substances 0.000 claims description 5
- 238000011068 loading method Methods 0.000 claims description 4
- 238000002447 crystallographic data Methods 0.000 claims description 3
- 230000005469 synchrotron radiation Effects 0.000 claims description 3
- 238000009792 diffusion process Methods 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 14
- 229920000106 Liquid crystal polymer Polymers 0.000 description 12
- 239000004977 Liquid-crystal polymers (LCPs) Substances 0.000 description 12
- 229920003223 poly(pyromellitimide-1,4-diphenyl ether) Polymers 0.000 description 10
- 238000011161 development Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000007888 film coating Substances 0.000 description 3
- 238000009501 film coating Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D9/00—Crystallisation
- B01D9/02—Crystallisation from solutions
Abstract
The invention relates to an in-situ crystallization device and method, wherein the in-situ crystallization device comprises an in-situ main board, a first surface and a second surface, and a plurality of hollowed in-situ holes penetrate through the first surface and the second surface; an in-situ template which is matched with the outline of the in-situ hole and is placed into the in-situ hole from the first surface of the in-situ main board; and the second surface of the in-situ main board is detachably covered and fixed with a sitting-drop in-situ crystallization board or a hanging-drop in-situ crystallization board. The in-situ crystallization device and method can be used as an LCP plate, a sitting-drop plate and a hanging-drop plate, can simultaneously accommodate a plurality of in-situ sample plates, can be directly used as an LCP crystallization experiment, can be assembled with the sitting-drop in-situ crystallization plate to be used as a sitting-drop crystallization experiment, and can be assembled with the hanging-drop in-situ crystallization plate to be used as a hanging-drop crystallization experiment, so that the problem of single protein crystallization mode in the prior art is solved.
Description
Technical Field
The invention relates to the field of protein crystal structure analysis, in particular to an in-situ crystallization device and method.
Background
Along with the development of structural biology, in-situ technology development has become more and more popular, namely, screening, crystallization and loading of crystals are carried out at the same position without artificial treatment on the crystals, and the method greatly simplifies the step of analyzing the crystal structure and avoids the interference of artificial factors, so that the method is suitable for analyzing the structures of tiny crystals and fragile crystals.
The most common mode in the in-situ technology is the in-situ plate technology which uses a low background scattering film and a bracket as a combination, in recent years, the in-situ plate device mainly comprises an in-situ small plate (i.e. an in-situ upper sample plate) and an in-situ large plate, the in-situ small plate is used based on a side angle head of a diffractometer, and the in-situ large plate is used based on a motor special for a line station, and the two plates have advantages and disadvantages. Recently, related researches begin to put forward a compatible in-situ plate concept, namely, the device integrates the advantages of an in-situ small plate and an in-situ large plate, the device can be used as an in-situ large plate, the device can also be independently disassembled to be used as an in-situ small plate, the assembly mode adopts push-pull mode, and the exploratory development is carried out on an assembly compatible crystallization plate suitable for a sitting-drop crystallization method, the compatible crystallization plate is a crystallization plate with reserved side holes, the in-situ small plate is inserted into the crystallization plate through the reserved side holes to form a closed crystallization space, during assembly, a single-sided film coating is needed to be carried out on the in-situ small plate by using the film coating plate, then the film coating plate is assembled with a baffle plate, and then tens of in-situ small plates are pushed into the side holes one by one, after crystals are found, the in-situ small plates are needed to be taken out one by using through plates or tweezers, the assembly mode is very tedious, and the air tightness is slightly insufficient compared with the traditional crystallization plate; the push-pull assembly is not suitable for the operation of a single in-situ small plate, and the efficiency is lower than that of the traditional crystallization plate; in addition, the compatible crystallization plate can only be used for protein sitting-drop crystallization, and the crystallization mode is single.
Disclosure of Invention
The invention aims to provide an in-situ crystallization device and method, which can solve the problems of single protein crystallization mode and low assembly efficiency.
In one aspect, the present invention provides an in situ crystallization apparatus comprising:
the in-situ mainboard is provided with a first surface and a second surface, and a plurality of hollowed-out in-situ holes penetrate through the first surface and the second surface;
an in-situ template which is matched with the outline of the in-situ hole and is placed into the in-situ hole from the first surface of the in-situ main board;
and the second surface of the in-situ main board is detachably covered and fixed with a sitting-drop in-situ crystallization board or a hanging-drop in-situ crystallization board.
Further, the in-situ sample plate is provided with at least one hollowed sample application hole and a trapezoid end positioned at the end part.
Further, the hole wall of the in-situ hole is provided with a protruding column and a small groove arranged along the circumference.
Further, one surface of the in-situ upper sample plate accommodated in the in-situ hole is supported on the protruding column, and the other surface of the in-situ upper sample plate and the small groove are positioned on the same horizontal plane.
Further, the second surface of the in-situ motherboard has a structure with complementary shapes with the sitting-drop in-situ crystallization board and the hanging-drop in-situ crystallization board.
Further, the complementary structures are buckles and clamping grooves or conical grooves and conical protrusions which are arranged in a crisscross mode.
Further, the sitting-drop in-situ crystallization plate is provided with a plurality of pool liquid holes and hollowed-out crystallization holes adjacent to the pool liquid holes, and each two Chi Yekong of the sitting-drop in-situ crystallization plate are matched with one in-situ hole of the in-situ main plate to form two closed crystallization chambers.
Further, the hanging drop in-situ crystallization plate is provided with a plurality of hollowed pool liquid holes, and every two Chi Yekong holes are matched with one in-situ hole to form two closed crystallization chambers.
Further, the in-situ holes are arranged in a matrix.
In another aspect of the present invention, an in situ crystallization method is provided, comprising the steps of:
s1: placing a plurality of in-situ upper templates in the in-situ holes of the in-situ main board;
s2: sealing the small groove of the in-situ motherboard with a film with low background scattering;
s3: selecting one of a fat cubic phase method, a sitting drop method and a hanging drop method for crystallization; when the fat cube phase method is selected, firstly cutting along the small groove of each in-situ hole of the in-situ upper sample plate by using a knife, turning over the in-situ upper sample plate with single-sided coating film, enabling one surface of the in-situ upper sample plate with the film to be supported by the protruding columns on the side arms of the in-situ holes of the in-situ main board, and then executing the step S4; when the sitting-drop method or the hanging-drop method is selected, directly executing the step S4;
s4: carrying out automatic sample application by using a liquid sample application machine;
s5: after sample application is completed, assembling to form a closed crystallization chamber; for fat cube phase method, the small groove of the in-situ main board is directly sealed by a film with low background scattering; for the sitting-drop method, mother liquor is added into a pool liquor hole of a sitting-drop in-situ crystallization plate, the sitting-drop in-situ crystallization plate is mounted on an in-situ main plate, and the opposite surface of the sitting-drop in-situ crystallization plate far from the in-situ main plate is sealed by using a transparent adhesive tape; for the hanging drop method, sealing one surface of the hanging drop in-situ crystallization plate far away from the in-situ main plate by using a transparent adhesive tape, adding mother liquor into a pool liquor hole of the hanging drop in-situ crystallization plate, and then installing the hanging drop in-situ crystallization plate to the in-situ main plate;
s6: after a period of cultivation, observing the growth condition of the crystal and obtaining an in-situ template of the double-sided coating film of the grown crystal; for the fat cube phase method, a knife is used for cutting along a small groove of an in-situ hole of the in-situ upper sample plate, so as to obtain the in-situ upper sample plate with double-sided coating; for the sitting-drop method and the hanging-drop method, cutting along the small groove of the in-situ hole of the in-situ upper template by using a knife, and turning over the in-situ upper template with a single-sided coating film, so that one surface of the in-situ upper template with a film is supported by the protruding columns on the side wall of the in-situ hole of the in-situ main board, sealing the film-free surface of the in-situ upper template by using a film with low background scattering again, and cutting along the small groove of the in-situ hole by using the knife again, thereby obtaining the in-situ upper template with a double-sided coating film;
s7: and fixing the in-situ upper template with the double-sided coating film on a magnetic base, placing the magnetic base in a crystal sample box, automatically loading samples at a synchrotron radiation beam line station, and finally collecting in-situ diffraction data.
The in-situ crystallization device and the method adopt the in-situ main board, can be used as an LCP board, a sitting-drop board and a hanging-drop board, can simultaneously accommodate a plurality of in-situ upper sample boards, can directly carry out LCP crystallization experiments according to requirements, can be assembled with the sitting-drop in-situ crystallization board for sitting-drop crystallization, and can also be assembled with the hanging-drop in-situ crystallization board for hanging-drop crystallization, so that the problem of single protein crystallization mode in the prior art is solved, and the in-situ crystallization device is convenient to assemble and better in sealing property through cover-type assembly, so that the crystals can obtain longer preservation time; the device can conveniently and directly operate the in-situ upper sample plate, and the special coating device is not required to be used for coating before operation, but in-situ coating can be performed, so that the working efficiency is improved relative to the prior device.
Drawings
FIG. 1 is a schematic view showing the structure of an in-situ crystallization apparatus according to a first embodiment of the present invention;
FIG. 2 is a schematic diagram of an in-situ master plate according to a first embodiment of the present invention;
fig. 3 is a schematic structural view of an in-situ motherboard according to a first embodiment of the present invention;
FIG. 4 is an enlarged view of a portion of the in-situ motherboard of FIG. 3;
FIG. 5 is a schematic structural view of an in-situ crystallization apparatus according to a second embodiment of the present invention;
FIG. 6 is a schematic view showing the structure of a sitting-drop in-situ crystallization plate according to a second embodiment of the present invention;
FIG. 7 is a schematic view showing the structure of the other surface of the sitting-drop in-situ crystallization plate according to the second embodiment of the present invention;
FIG. 8 is a schematic view of a structure of a side of an in-situ motherboard away from a small groove according to a second embodiment of the present invention;
FIG. 9 is a schematic structural view of an in-situ crystallization apparatus according to a third embodiment of the present invention;
FIG. 10 is a schematic view of a structure of a hanging-drop in-situ crystallization plate according to a third embodiment of the present invention;
fig. 11 is a flow chart of an in situ crystallization method according to a fourth embodiment of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.
In the description of the present invention, it should be understood that the terms "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like indicate or are based on the orientation or positional relationship shown in the drawings, merely to facilitate description of the invention and to simplify the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the invention.
As shown in fig. 1, an embodiment of the present invention provides an in-situ crystallization device, which includes an in-situ motherboard 20 and a plurality of in-situ templates 10, wherein the in-situ motherboard 20 is provided with a plurality of hollowed in-situ holes 21, and the in-situ templates 10 are accommodated in the in-situ holes 21 from one surface (i.e. a first surface) of the in-situ motherboard 20.
As shown in fig. 2, the in-situ sample plate 10 has at least one hollowed sample application hole 11 and a trapezoid end 12 at the end, the sample application can be performed through the sample application hole 11, the trapezoid end 12 is adapted to be matched with a magnetic base, and after the crystallization is completed and the in-situ sample plate 10 is taken out from the in-situ hole 21, the trapezoid end 12 can be assembled with the magnetic base to be mounted on a side corner of a diffractometer. The magnetic base and diffractometer are devices commonly used in crystallization processes, and their structures are well known in the art and will not be described here.
As shown in fig. 3 and 4, the in-situ hole 21 is provided with a small groove 22 arranged along the circumference thereof, the hole wall of the in-situ hole 21 is provided with a protruding column 23 for supporting the in-situ upper sample plate, and when one surface of the in-situ upper sample plate is contacted with the protruding column 23 of the hollowed-out in-situ hole 21 and is supported, the other surface of the in-situ upper sample plate is positioned on the same plane with the small groove 22; in the crystallization process, after the crystals are found, the coated in-situ master plate 10 is obtained by cutting along the small grooves 22 with a knife.
The in-situ holes 21 may be arranged in a matrix, and the number thereof may be set as needed, which is not limited in the present invention.
Through the in-situ template 10 and the in-situ main board 20, the crystallization experiment of the fat cubic phase method (Lipid Cubic Phase method, LCP) can be completed, and the specific process is as follows:
firstly, placing the in-situ master plate 10 in the in-situ holes 21 of the in-situ main plate 20, then sealing the small grooves 22 of the in-situ main plate 20 by using a film with low background scattering such as Kapton film, then cutting along the small grooves of each in-situ hole 21 by using a knife, and turning over the in-situ master plate 10 with single-sided coating so that the surface with the film is supported by the protruding columns 23 on the side walls of the in-situ holes 21; and then, carrying out automatic sample application by using a liquid sample application machine, directly sealing the small groove 22 of the in-situ main board 20 by using a film with low background scattering, such as a Kapton film, forming a closed crystallization chamber, after a period of culture, observing the crystal growth condition in the in-situ upper sample board 10, selecting the in-situ upper sample board 10 with the grown crystal, cutting along the small groove 22 by using a knife, and obtaining the in-situ upper sample board 10 with double-sided coating, thereby completing the crystallization experiment of the LCP method.
As shown in fig. 5 and 6, on the basis of the first embodiment, the in-situ crystallization device of the second embodiment further includes a sitting-drop in-situ crystallization plate 30, on which a plurality of non-hollowed-out pond liquid holes 31 and hollowed-out crystallization holes 32 adjacent to the pond liquid holes 31 are formed, wherein the crystallization holes 32 are used for providing a gas-phase diffusion space for protein crystallization, and at the same time, the crystals can be ensured to be directly observed under a microscope; the sitting-drop in-situ crystallization plate 30 is detachably covered on one surface (i.e. the second surface) of the in-situ main plate 20 away from the in-situ upper template 10, and after the covering, each in-situ hole 21 of the in-situ main plate 20 is matched with two pool liquid holes 31 to form two closed crystallization chambers.
As shown in fig. 7 and 8, the surface of the in-situ master plate 20 away from the in-situ top plate 10 and the bottom surface of the sitting-drop in-situ crystallization plate 30 have a complementary structure, so as to facilitate rapid positioning and combination of the two. For example, the in-situ motherboard 20 is provided with criss-cross conical grooves 24 (or conical protrusions), the sitting-drop in-situ crystallization board 30 is provided with conical protrusions 33 (or conical grooves) matched with the conical grooves 24 (or conical protrusions), and the conical protrusions 33 are inserted into the conical grooves 24 so as to cover the two. It is conceivable that the complementary structure may also be a catch and a catch groove, the catch being snapped into the catch groove, thereby achieving a fixation between the two plates.
The sitting-drop in-situ crystallization plate 30 is provided with a base 34, a conical bulge 33 and other structures are arranged on the base 34, and when sample application is carried out in a sitting-drop crystallization experiment, the in-situ crystallization device can be clamped into a groove of a sample application machine through the base 34, so that automatic sample application is realized.
The in-situ crystallization device of the second embodiment can realize a sitting-drop crystallization experiment, and the specific process is as follows:
firstly, placing a plurality of in-situ sample plates 10 in an in-situ hole 21 of an in-situ main plate 20, sealing a small groove surface of the in-situ main plate 20 by using a film with low background scattering, such as a Kapton film, then automatically spotting by using a liquid spotting machine, adding mother liquor into a pool liquor hole 31 of a sitting-drop in-situ crystallization plate 30 after spotting is finished, then installing the sitting-drop in-situ crystallization plate 30 on the in-situ main plate 20, and sealing the opposite surface of the sitting-drop in-situ crystallization plate 30 far from the in-situ main plate 20 by using a transparent adhesive tape to form a closed crystallization chamber; after a period of cultivation, the growth condition of the crystal is observed, the in-situ upper template 10 for growing the crystal is selected, specifically, a knife is used for cutting along the small groove 22, the in-situ upper template 10 with a single-sided coating film is turned over, one surface of the in-situ upper template 10 with the film is supported by the protruding column 23 on the side wall of the hollowed-out in-situ hole 21 of the in-situ main board, the film-free surface of the in-situ upper template 10 is sealed again by a film with low background scattering such as Kapton film, the knife is used for cutting again along the small groove 22 of the hollowed-out in-situ hole, and the in-situ upper template 10 with a double-sided coating film can be obtained, so that the crystallization experiment by the sitting-drop method is completed.
The pool liquid holes 31 may be arranged in a matrix, and the number thereof may be set as needed, which is not limited in the present invention.
In this example, chi Yekong and crystallization holes 32 are 8×12, and in-situ holes 21 are 4×12.
As shown in fig. 9 and 10, on the basis of the first embodiment, the in-situ crystallization device of the third embodiment further includes a hanging-drop in-situ crystallization plate 40, which has a plurality of hollow pool liquid holes 41, and the hanging-drop in-situ crystallization plate 40 is detachably covered on the in-situ main plate 20, and after the covering, each in-situ hole 21 of the in-situ main plate 20 is matched with two pool liquid holes 41 to form two closed crystallization chambers.
The hanging-drop in-situ crystallization plate 40 is also provided with a structure complementary to the side of the in-situ motherboard 20 remote from the cuvette 22. For example, when crisscrossed tapered grooves 24 are provided on the in-situ motherboard 20, tapered protrusions 42 are provided on the hanging-drop in-situ crystallization board 40, and the tapered protrusions 42 are inserted into the tapered grooves 24 so as to cover the two. It is conceivable that the complementary structure may also be a catch and a catch groove, the catch being snapped into the catch groove, thereby achieving a fixation between the two plates.
Chi Yekong can be arranged in a matrix, and the number of the Chi Yekong can be set according to the requirement, which is not limited in the invention.
In this embodiment, chi Yekong has 8×12 holes and in-situ holes 21 have 4×12 holes.
The in-situ crystallization device of the third embodiment can realize a hanging drop crystallization experiment, and the specific process is as follows:
firstly, placing a plurality of in-situ upper plates 10 in an in-situ hole 21 of an in-situ main plate 20, sealing a small groove surface of the in-situ main plate 20 by using a film with low background scattering, such as a Kapton film, then automatically spotting by using a liquid spotting machine, sealing one surface of a hanging drop in-situ crystallization plate 40 far away from the in-situ main plate 20 by using a transparent adhesive tape after spotting is finished, adding mother liquor into a pool liquid hole 41, and then installing the hanging drop in-situ crystallization plate 40 to the in-situ main plate 20 to form a closed crystallization chamber; after a period of cultivation, the crystal growth condition is observed, the in-situ upper template 10 for growing the crystal is selected, specifically, a knife is used for cutting along the small groove 22, the in-situ upper template 10 with a single-sided coating film is turned over, one surface of the in-situ upper template 10 with the film is supported by the protruding column 23 on the side wall of the hollowed-out in-situ hole 21 of the in-situ main board, the film-free surface of the in-situ upper template 10 is sealed again by a film with low background scattering such as Kapton film, and the knife is used for cutting again along the small groove 22 of the hollowed-out in-situ hole, so that the in-situ upper template 10 with a double-sided coating film can be obtained, and the hanging drop crystallization experiment is completed.
The in-situ crystallization device provided by the embodiment of the invention can be used as an LCP plate, a sitting-drop plate and a hanging-drop plate, and simultaneously accommodates a plurality of in-situ sample plates 10, can be directly used as an LCP crystallization experiment, can be assembled with the sitting-drop in-situ crystallization plate 30 to be used as a sitting-drop crystallization experiment, and can be assembled with the hanging-drop in-situ crystallization plate 40 to be used as a hanging-drop crystallization experiment, so that the problem of single protein crystallization mode in the prior art is solved, and the in-situ crystallization device is assembled by a cover-type, is convenient to assemble and has better sealing property, so that crystals can be stored for a longer time; the device can conveniently and directly operate the in-situ upper template 10, and the in-situ coating can be performed without using a special coating device before operation, so that the efficiency is improved compared with the prior device.
A fourth embodiment of the present invention provides an in situ crystallization method, comprising the steps of:
s1: placing a plurality of in-situ sample plates 10 in the in-situ holes 21 of the in-situ main plate 20, wherein the in-situ sample plates 10 are supported by the protruding columns 23 on the side walls of the in-situ holes 21;
s2: sealing the small grooves 22 of the in-situ master 20 with a film having low background scattering, such as Kapton film;
s3: selecting one of LCP method, sitting method and hanging drop method for crystallization; when the LCP method is selected, a knife is firstly used for cutting along the small groove 22 of each in-situ hole 21, and the in-situ upper template 10 with single-sided coating is turned over, so that the surface of the in-situ upper template 10 with the film is supported by the protruding columns 23 on the side arms of the in-situ holes 21 of the in-situ main board 20, and then the step S4 is executed; when the sitting-drop method or the hanging-drop method is selected, directly executing the step S4;
s4: carrying out automatic sample application by using a liquid sample application machine;
s5: after sample application is completed, assembling to form a closed crystallization chamber;
for LCP method: the small grooves 22 of the in-situ master are sealed directly with a film with low background scattering, such as Kapton film.
For the sitting-drop method: mother liquor is added into a pool liquid hole 31 of the sitting-drop in-situ crystallization plate 30, the sitting-drop in-situ crystallization plate 30 is mounted on the in-situ main plate 20, and the opposite surface of the sitting-drop in-situ crystallization plate 30 away from the in-situ main plate 20 is sealed by using transparent adhesive tape.
For the hanging drop method: the side of the hanging-drop in-situ crystallization plate 40 away from the in-situ motherboard 20 is sealed with a transparent adhesive tape, mother liquor is added into the pool liquor hole 41, and then the hanging-drop in-situ crystallization plate 40 is mounted on the in-situ motherboard 20.
S6: after a period of cultivation, observing the growth condition of the crystal and obtaining an in-situ upper template 10 with double-sided coating films of the grown crystal;
for LCP method: the in-situ master 10 with the double-sided coating was obtained by cutting along the small groove 22 with a knife.
For the sitting-drop method: cutting along the small groove 22 by a knife, turning over the in-situ template 10 with a single-sided coating, so that the surface of the in-situ template 10 with the film is supported by the protruding columns 23 on the side wall of the hollowed-out in-situ hole 21 of the in-situ main board, sealing the non-film surface of the in-situ template 10 by a film with low background scattering such as Kapton film, and cutting again along the small groove 22 of the hollowed-out in-situ hole by the knife, thereby obtaining the in-situ template 10 with double-sided coating.
For the hanging drop method: cutting along the small groove 22 by a knife, turning over the in-situ upper template 10 with a single-sided coating film, supporting the surface of the in-situ upper template 10 with the film by the protruding column 23 on the side wall of the hollowed-out in-situ hole 21 of the in-situ main board, sealing the non-film surface of the in-situ upper template 10 by a film with low background scattering such as Kapton film, and cutting again along the small groove 22 of the hollowed-out in-situ hole by the knife to obtain the in-situ upper template 10 with double-sided coating film.
S7: and fixing the in-situ upper template with the double-sided coating film on a magnetic base, placing the magnetic base in a crystal sample box, automatically loading samples at a synchrotron radiation beam line station, and finally collecting in-situ diffraction data.
The in-situ crystallization method provided by the embodiment of the invention can be used for performing LCP (liquid crystal polymer) crystallization, sitting-drop crystallization or hanging-drop crystallization according to the needs, and solves the problem of single protein crystallization mode in the prior art.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications can be made to the above-described embodiment of the present invention. All simple, equivalent changes and modifications made in accordance with the claims and the specification of this application fall within the scope of the patent claims. The present invention is not described in detail in the conventional art.
Claims (5)
1. An in situ crystallization apparatus, comprising:
the in-situ mainboard is provided with a first surface and a second surface, and a plurality of hollowed-out in-situ holes penetrate through the first surface and the second surface; the hole wall of the in-situ hole is provided with a protruding column and a small groove arranged along the circumference;
an in-situ template which is matched with the outline of the in-situ hole and is placed into the in-situ hole from the first surface of the in-situ main board; one surface of the in-situ upper sample plate accommodated in the in-situ hole is supported on the protruding column, and the other surface of the in-situ upper sample plate and the small groove are positioned on the same horizontal plane;
a sitting-drop in-situ crystallization plate or a hanging-drop in-situ crystallization plate is detachably covered and fixed on the second surface of the in-situ main plate;
the in-situ upper sample plate is provided with at least one hollowed sample application hole and a trapezoid end positioned at the end part;
the sitting-drop in-situ crystallization plate is provided with a plurality of non-hollowed-out pool liquid holes and hollowed-out crystallization holes adjacent to the non-hollowed-out pool liquid holes, each two non-hollowed-out pool liquid holes of the sitting-drop in-situ crystallization plate are matched with one in-situ hole of the in-situ main plate to form two closed crystallization chambers, and the hollowed-out crystallization holes are used for providing a gas phase diffusion space of protein crystallization and enabling the crystals to be directly observed under a microscope;
the hanging drop in-situ crystallization plate is provided with a plurality of hollowed-out pool liquid holes, and every two hollowed-out pool liquid holes are matched with one in-situ hole to form two closed crystallization chambers.
2. The in situ crystallization apparatus of claim 1, wherein the second surface of the in situ motherboard has a complementary shape configuration to the sitting-drop in situ crystallization plate and hanging-drop in situ crystallization plate.
3. The in-situ crystallization apparatus according to claim 2, wherein the complementary shaped structures are buckles and clamping grooves or the complementary shaped structures are conical grooves and conical protrusions arranged in a crisscross manner.
4. The in situ crystallization apparatus according to claim 1, wherein the in situ pores are arranged in a matrix.
5. An in situ crystallization method, characterized in that it is carried out with the in situ crystallization device according to claim 1 and comprises the steps of:
s1: placing a plurality of in-situ upper templates in the in-situ holes of the in-situ main board;
s2: sealing the small groove of the in-situ motherboard with a film with low background scattering;
s3: selecting one of a fat cubic phase method, a sitting drop method and a hanging drop method for crystallization; when the fat cube phase method is selected, firstly cutting along the small groove of each in-situ hole of the in-situ upper sample plate by using a knife, turning over the in-situ upper sample plate with single-sided coating film, enabling one surface of the in-situ upper sample plate with the film to be supported by the protruding columns on the side arms of the in-situ holes of the in-situ main board, and then executing the step S4; when the sitting-drop method or the hanging-drop method is selected, directly executing the step S4;
s4: carrying out automatic sample application by using a liquid sample application machine;
s5: after sample application is completed, assembling to form a closed crystallization chamber; for fat cube phase method, the small groove of the in-situ main board is directly sealed by a film with low background scattering; for the sitting-drop method, mother liquor is added into a pool liquor hole of a sitting-drop in-situ crystallization plate, the sitting-drop in-situ crystallization plate is mounted on an in-situ main plate, and the opposite surface of the sitting-drop in-situ crystallization plate far from the in-situ main plate is sealed by using a transparent adhesive tape; for the hanging drop method, sealing one surface of the hanging drop in-situ crystallization plate far away from the in-situ main plate by using a transparent adhesive tape, adding mother liquor into a pool liquor hole of the hanging drop in-situ crystallization plate, and then installing the hanging drop in-situ crystallization plate to the in-situ main plate;
s6: after the culture, observing the growth condition of the crystal and obtaining an in-situ upper template of the double-sided coating film of the grown crystal; for the fat cube phase method, a knife is used for cutting along a small groove of an in-situ hole of the in-situ upper sample plate, so as to obtain the in-situ upper sample plate with double-sided coating; for the sitting-drop method and the hanging-drop method, cutting along the small groove of the in-situ hole of the in-situ upper template by using a knife, and turning over the in-situ upper template with a single-sided coating film, so that one surface of the in-situ upper template with a film is supported by the protruding columns on the side wall of the in-situ hole of the in-situ main board, sealing the film-free surface of the in-situ upper template by using a film with low background scattering again, and cutting along the small groove of the in-situ hole by using the knife again, thereby obtaining the in-situ upper template with a double-sided coating film;
s7: and fixing the in-situ upper template with the double-sided coating film on a magnetic base, placing the magnetic base in a crystal sample box, automatically loading samples at a synchrotron radiation beam line station, and finally collecting in-situ diffraction data.
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|---|---|---|---|---|
| GB0319039D0 (en) * | 2002-08-29 | 2003-09-17 | Ekisho Sentan Kk | Method of in-situ monitoring of crystallisation state |
| US7498229B1 (en) * | 2005-02-09 | 2009-03-03 | Translucent, Inc. | Transistor and in-situ fabrication process |
| CN111855718A (en) * | 2020-07-29 | 2020-10-30 | 中国科学院上海高等研究院 | Protein crystal and crystal in-situ diffraction data acquisition device and acquisition method thereof |
| CN113376191A (en) * | 2021-06-08 | 2021-09-10 | 中国科学院上海应用物理研究所 | In-situ-based device and method for high-throughput crystal culture and rapid sample loading |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0319039D0 (en) * | 2002-08-29 | 2003-09-17 | Ekisho Sentan Kk | Method of in-situ monitoring of crystallisation state |
| US7498229B1 (en) * | 2005-02-09 | 2009-03-03 | Translucent, Inc. | Transistor and in-situ fabrication process |
| CN111855718A (en) * | 2020-07-29 | 2020-10-30 | 中国科学院上海高等研究院 | Protein crystal and crystal in-situ diffraction data acquisition device and acquisition method thereof |
| CN113376191A (en) * | 2021-06-08 | 2021-09-10 | 中国科学院上海应用物理研究所 | In-situ-based device and method for high-throughput crystal culture and rapid sample loading |
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