CN113376191A - In-situ-based device and method for high-throughput crystal culture and rapid sample loading - Google Patents
In-situ-based device and method for high-throughput crystal culture and rapid sample loading Download PDFInfo
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- CN113376191A CN113376191A CN202110638377.XA CN202110638377A CN113376191A CN 113376191 A CN113376191 A CN 113376191A CN 202110638377 A CN202110638377 A CN 202110638377A CN 113376191 A CN113376191 A CN 113376191A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
- G01N23/20008—Constructional details of analysers, e.g. characterised by X-ray source, detector or optical system; Accessories therefor; Preparing specimens therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
- G01N23/20008—Constructional details of analysers, e.g. characterised by X-ray source, detector or optical system; Accessories therefor; Preparing specimens therefor
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Abstract
The invention provides a device and a method for high-throughput culture and rapid sample loading of crystals based on in-situ, which comprises the following steps: the in-situ upper sample plates are provided with hollowed sample application holes; the coating plate is used for providing a coating place and is provided with a plurality of first coating grooves and a plurality of second coating grooves, each first coating groove has the size equivalent to the in-situ sample feeding plate, and each second coating groove has the length which is multiple times of the length of the in-situ sample feeding plate and the same width and is used for accommodating a plurality of in-situ sample feeding plates simultaneously; the in-situ crystallization plate for crystal culture comprises a plurality of pool liquid holes and a plurality of side holes which penetrate through the pool liquid holes in the front and back directions, wherein the side holes are positioned above the pool liquid holes; and a through plate adapted to the side hole in the in situ crystallization plate. According to the invention, the in-situ sample loading plate matched with the in-situ crystallization plate, the coating plate matched with the in-situ sample loading plate and convenient for coating and cutting, and the through plate matched with the in-situ crystallization plate are utilized, so that the optimization, high-flux culture and rapid sample loading of crystals on the premise of in-situ are realized.
Description
Technical Field
The invention relates to the field of protein crystal structure analysis, in particular to an in-situ based device and method for high-throughput crystal culture and rapid sample loading.
Background
With the development of structural biology, how to efficiently and quickly develop protein crystal culture, screening optimization and sample loading becomes more and more important, the development of an in-situ technology realizes the continuous collection of diffraction data of crystals in the growth environment, the process of transferring samples is omitted, the sample loading efficiency of the crystals is greatly improved, and the method is particularly suitable for the diffraction data acquisition of micro crystals or fragile crystals at normal and low temperatures.
The in-situ method is generally realized by two in-situ plates, one is a special in-situ large plate for the beam line station, but the in-situ large plate has an oversize size compared with the conventional nylon sample loading ring, and other hardware systems such as motors and the like are required to be added or changed for matching in the in-situ diffraction data acquisition process. Secondly, the existing in-situ plate cannot perform quick centering based on rotation of an angle measuring instrument like a conventional nylon sample loading ring, namely, crystals are quickly transferred to a light path, and motor parameters are directly changed to move the in-situ plate, so that the operation complexity is increased and certain potential safety hazards are caused; the other type of in-situ plate is an in-situ small plate used for the beam line station, a motor and other hardware systems of the beam line station are not required to be added or changed for cooperation, a conventional crystal sample loading mode can be adopted for diffraction data acquisition, but the in-situ small plate is small in size, high in crystal culture operation difficulty and low in sample loading efficiency due to the adoption of a manual sample loading mode, and application of the in-situ small plate is greatly limited. With the wider application of in-situ technology, how to conveniently culture crystals and improve the efficiency of crystal sample loading becomes more and more important, and the development of related hardware and software is continuously developed.
Disclosure of Invention
The invention aims to provide an in-situ-based device and method for high-throughput culture and rapid sample loading of crystals, so as to solve the problems of difficulty in-situ platelet crystal culture and low sample loading efficiency in the prior art.
According to a first aspect of the present invention, there is provided an in situ-based device for high throughput culture and rapid loading of crystals, comprising: the in-situ upper sample plates are provided with hollow sample application holes; the coating plate is used for providing a coating place and is provided with a plurality of first coating grooves and a plurality of second coating grooves, each first coating groove has a size equivalent to that of the in-situ sample loading plate, and each second coating groove has a length which is several times that of the in-situ sample loading plate and an equal width and is used for accommodating a plurality of in-situ sample loading plates simultaneously; the in-situ crystallization plate is used for crystal culture and comprises a plurality of pool liquid holes, the side surface of the in-situ crystallization plate also comprises a plurality of side holes which penetrate through the side surface of the in-situ crystallization plate from front to back, and the side holes are positioned above the pool liquid holes; and a through plate, wherein the size of the through plate is matched with the size of the side hole on the in-situ crystallization plate; when the plated film plate and the in-situ crystallization plate are placed side by side on the same horizontal plane, a first plated film groove or a second plated film groove on the plated film plate is aligned with a side hole on the in-situ crystallization plate, the in-situ sample loading plate placed in the first plated film groove can enter the in-situ crystallization plate through the side hole, at least one point sample hole on the in-situ sample loading plate and one pool liquid hole of the in-situ crystallization plate form a crystallization chamber, and therefore high-throughput crystal culture and rapid sample loading based on the in-situ are achieved.
According to a preferred embodiment of the present invention, the in-situ crystallization plate has a matrix arrangement of liquid wells, and the number of the liquid wells is x × y.
Preferably, the apparatus further comprises a detachable baffle plate, the upper central position of the in-situ crystallization plate is provided with a groove extending along the whole length direction, the size of the baffle plate is matched with the groove, and the crystallization chamber adjacent to the baffle plate after assembly can form a closed gas phase diffusion space.
In designing the device of the invention, the length of the in-situ sample plate is limited by the hardware phase of the wire station, so the length cannot be too long, the useless space on the in-situ sample plate is reduced as much as possible to increase the pore volume of the sample application, the size of the crystallization plate which can be used on the common automatic crystal sample application machine is larger, and a large amount of in-situ sample plates are needed to fill the crystallization plate in order to meet the requirements of the wire station and fully utilize the automatic crystal sample application machine. Thus, two problems occur, one is that the crystallization plate has four in-situ upper sample plates in a row, and it is difficult to correct the positions of the four plates at a time, because the plate processing has errors, and the subsequent observation can be influenced as long as the in-situ upper sample plates are slightly deviated; secondly, if no baffle is provided, the front and rear side holes of the crystallization plate are communicated, the size standard of the in-situ sample loading plate is fixed, four in-situ sample loading plates which are identical are inserted, and the difficulty that the sample dispensing holes in each in-situ sample loading plate correspond to the crystallization chamber is high. The invention can meet the requirements of both aspects by designing the baffle plate.
It can be seen that the baffle is most suitably arranged at the middle position of the in-situ crystallization plate in the width direction, using the conventional automatic spotting machine and the Shanghai light source individual line station.
Preferably, the in situ crystallization plate is adapted to an automated spotting machine.
Preferably, the small end of the in-situ upper template has a guide slope to facilitate entry into the side hole.
Preferably, the film plating plate and the in-situ crystallization plate can be spliced on the same horizontal plane, and the side surface of the film plating plate and the side surface of the in-situ crystallization plate have structures with complementary shapes, so that the stable assembly of the film plating plate and the in-situ crystallization plate is realized. Preferably, the bottom plates of the film plating plate and the in-situ crystallization plate can be assembled, and the height of the film plating plate is the same as that of the side hole clamping groove of the in-situ crystallization plate, so that the in-situ upper template can be placed in the in-situ crystallization plate in batch, the in-situ upper template can be taken out from the in-situ crystallization plate in batch, and the film can be plated on the film plating plate in batch.
Preferably, the first and second coating grooves have first and second scores arranged along at least a part of the circumferential direction, respectively, so as to facilitate cutting during coating.
Preferably, the number of the second coating grooves is matched with the number of the side holes of the in-situ crystallization plate, so that the mass assembly or disassembly of the in-situ template and the in-situ crystallization plate in a column is facilitated.
Preferably, the number of the side hole clamping grooves of the in-situ crystallization plate is set according to the size of a common crystallization plate of an automatic sample applicator, so that the phase correction of the motor is facilitated.
Preferably, the length and width of the through plate are matched with the size of the side holes, so that the in-situ template in the in-situ crystallization plate can be pushed out to the coated plate in batches in a row or a column.
Preferably, the in-situ crystallization plate is internally provided with a hollow clamping groove device in the side hole, so that the in-situ upper sample plate is convenient to assemble, and the growth condition of the crystal is convenient to observe under a microscope.
According to a second aspect of the present invention, there is provided an in situ based method for high throughput culture and rapid loading of crystals, the method comprising the steps of: s1: providing an in situ based crystal high throughput culturing and rapid loading device as described above; s2: placing the in-situ sample loading plate in a first film coating groove on the film coating plate for back film coating, and pushing the turned in-situ sample loading plate into the in-situ crystallization plate through the side hole; s3: carrying out sample application operation on the in-situ crystallization plate by using an automatic sample application machine, and then sealing the whole in-situ crystallization plate to form a plurality of closed crystallization chambers for carrying out protein crystallization; s4: pushing the in-situ upper sample plates out of the in-situ crystallization plate to second coating grooves on the coating plate in batches by using a through plate, wherein each second coating groove contains a plurality of in-situ upper sample plates simultaneously, and coating the front surface of each second coating groove; s5: and finally, fixing the in-situ upper template with the double-sided coated film on a magnetic base, and placing the template into a crystal sample box, so that automatic sample loading can be performed at a synchrotron radiation beam line station.
According to a preferred embodiment of the present invention, step S2 includes: sealing the in-situ sample loading plate by using a film with low background scattering property, and then cutting along a first nick on the first film coating groove by using a blade to obtain an in-situ sample loading plate with a single-sided film coating; step S4 further includes: and sealing the in-situ sample loading plate by using a film with low background scattering property, then cutting along a second nick on the second film coating groove by using a blade, and separating the adjacent in-situ sample loading plates to obtain the in-situ sample loading plate with double-sided coating.
According to a preferred embodiment of the present invention, step S2 further includes: and a detachable baffle is provided, a groove extending along the whole length direction is formed in the middle position above the in-situ crystallization plate, the baffle is inserted into the groove, and the in-situ upper sample plate with a single-side coated film can be pushed into the in-situ crystallization plate from two sides through the side hole, so that the in-situ upper sample plate and the in-situ crystallization plate can be accurately assembled.
According to the in-situ-based device for high-flux crystal culture and rapid sample loading, one surface of the in-situ sample loading plate is coated with a film in advance by using the shorter first film coating groove on the film coating plate, and when crystallization is completed, the other surfaces of all the in-situ sample loading plates are coated with films in batch by using the longer second film coating groove. The coating plate comprises a plurality of first coating grooves and a plurality of second coating grooves which are arranged in an aligned mode, first notches with shallow relative depths are further formed in the edges of the first coating grooves, after the in-situ upper sample plate is placed in the first coating grooves, the in-situ upper sample plate is sealed through a film with low background scattering, such as a polyimide film, and then cutting is conducted through a blade along the first notches, so that the in-situ upper sample plate with a single-side coating can be obtained. After the in-situ template is crystallized on the in-situ crystallization plate, a plurality of in-situ templates can be simultaneously contained in each second coating groove, the non-coated surface of the in-situ template is coated with a low background scattering film such as a polyimide film, and then the in-situ template with double-coated surfaces can be obtained by cutting along the second nicks.
According to the high-throughput crystal culture and rapid sample loading device provided by the invention, the high-throughput culture of the crystal is realized by utilizing the in-situ crystallization plate, the in-situ crystallization plate is provided with the top groove and the side hole clamping groove, the baffle plate can be assembled by the top groove and the in-situ crystallization plate, and the in-situ sample loading plate can be assembled by the side hole clamping groove and the in-situ crystallization plate. After the single-side coating of the in-situ upper template, firstly assembling a baffle plate with a top groove of an in-situ crystallization plate, then assembling the in-situ crystallization plate with a chassis of a coating plate, pushing the in-situ upper template coated with the single-side coating on the coating plate into the in-situ crystallization plate, repeating the process until the in-situ upper template is pushed to the baffle plate, carrying out the same steps on the other side of the in-situ crystallization plate where a side hole clamping groove is reserved, completing the assembly of the whole in-situ crystallization plate and the in-situ upper template, carrying out the sample application operation on the in-situ crystallization plate by using an automatic sample application machine, then sealing the whole in-situ crystallization plate to form a plurality of closed crystallization chambers, after protein is crystallized, taking down the baffle plate, pushing the in-situ upper template on the in-situ crystallization plate out on the coating plate by using a through plate, carrying out the double-side coating, finally fixing the in-situ upper template coated with double-sides on a magnetic base, and placing the in a crystal sample box uni-puck, the automated loading can be performed at the synchrotron radiation beam-line station.
According to the device and the method for high-throughput culture and rapid sample loading of the crystals, the advantages of in-situ diffraction can be fully utilized, in addition, an in-situ crystallization plate matched with an automatic sample applicator, an in-situ sample loading plate matched with the in-situ crystallization plate and a crystal sample box and a coating plate matched with the in-situ sample loading plate are developed, and the optimization, high-throughput culture and rapid sample loading of the crystals are realized on the premise of in-situ. By the method, the efficiency of protein structure analysis is greatly improved, and an important step is taken for realizing full-automatic protein structure analysis. The invention is suitable for the diffraction data acquisition of the crystal cultured by the sitting drop method, in particular to the micro crystal.
Drawings
FIG. 1 is an enlarged schematic view of a single in-situ upper template according to a preferred embodiment of the present invention;
FIG. 2 is a schematic structural view of a coated board according to a preferred embodiment of the present invention;
FIG. 3 is a schematic structural view of an in-situ crystallization plate according to a preferred embodiment of the present invention;
FIG. 4 is a schematic structural view of a baffle according to a preferred embodiment of the present invention;
FIG. 5 is a schematic structural view of a through plate according to a preferred embodiment of the present invention;
FIG. 6 is a schematic view of the coated plate and the in-situ crystallization plate being assembled on the same plane;
wherein the reference numerals are as follows: 10. in-situ template loading; 11. spotting the sample wells; 12. a small end; 20. coating a film plate; 21. a first coating tank; 22. a first score; 23. a second coating tank; 24. a second score; 25. a coated plate chassis; 30. crystallizing the plate in situ; 31. an in-situ crystallization plate chassis; 32. a side hole; 33. a top groove; 34. a pool liquid hole; 35. crystallizing the top of the plate in situ; 40. a baffle plate; 50. and (5) passing through the plate.
Detailed Description
The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
In the description of the present invention, it is to be understood that the terms "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like, as used herein, are used in an orientation or positional relationship indicated in the drawings for convenience in describing the present invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed in a particular orientation, and be operated in a particular manner, and are not to be construed as limiting the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
Referring to fig. 1 to 5, a high throughput crystal culturing and rapid loading device according to a preferred embodiment of the present invention comprises: the in-situ crystallization device comprises an in-situ upper template 10, a coating plate 20, an in-situ crystallization plate 30, a baffle plate 40 and a through plate 50.
As shown in FIG. 1, the in-situ upper template 10 has four wells 11 with an open hole and a small end 12 at one end, through which wells 11 the sample can be spotted, and the small end 12 has a guiding bevel as shown in the figure to facilitate the assembly into the in-situ crystallization plate 30. It should be understood, however, that the number of wells 11 in each home position upper template 10 is not limited to four.
As shown in fig. 2, the plating plate 20 has twelve first plating grooves 21 and twelve second plating grooves 23 aligned in the front-rear direction, the first plating grooves 21 correspond to the original position upper template 10 in size, and the second plating grooves 23 have lengths several times as long as the original position upper template 10 and equal widths. According to the preferred embodiment, the second plating bath 23 has a length four times that of the in-situ upper template 10, and thus a single second plating bath 23 can simultaneously accommodate four in-situ upper templates 10. It should be understood that the size of the second plating bath 23 may be determined according to the design of the in-situ crystallization plate 30, or according to the arrangement of the bath holes. The first and second plating grooves 21 and 23 also have first and second scores 22 and 24, respectively, arranged along three sides of the circumference, so as to facilitate cutting during plating.
As shown in FIG. 3, the in-situ crystallization plate 30 includes 96 (8X 12) well liquid holes 34, and the lateral surface of the in-situ crystallization plate 30 further includes twelve side holes 32 penetrating in the front-rear direction, which are located above the well liquid holes 34, and a top groove 33 extending along the entire length of the upper part of the in-situ crystallization plate 30 in the width direction. It should be understood that in the description of the present invention, the length direction is from left to right and the width direction is from front to back with reference to fig. 3.
As shown in fig. 4, the baffle 40 has a strip-shaped structure, and after the baffle 40 is inserted into the top groove 33, the in-situ upper template 10 with a single-sided coating can be pushed into the in-situ crystallization plate 30 through the side holes 32 at two sides, so that the in-situ upper template 10 and the in-situ crystallization plate 30 can be accurately assembled, and the problem that the in-situ upper template 10 cannot be accurately positioned during one-sided assembly is fundamentally avoided. At the same time, the baffle 40 is matched with the size of the top groove 33, so that the baffle 40 can form a closed space with the crystallization chambers at two ends of the top groove 33 after assembly, and a gas phase diffusion condition of protein crystals is provided.
As shown in FIG. 5, the through plate 50 also has a strip-shaped structure, and the size of the through plate 50 is adapted to the size of the side holes 32 on the in-situ crystallization plate 10, so that a row of four in-situ upper templates 10 in the in-situ crystallization plate 30 can be pushed out to the second coating grooves 23 of the coating plate 20 in batch.
Preferably, the coated plate 20 and the in-situ crystallization plate 30 can be spliced on the same horizontal plane, and the side surface of the coated plate 20 and the side surface of the in-situ crystallization plate 30 have a structure with a complementary shape. As shown in fig. 2, 3 and 6, the bottom plate 25 of the plated film plate and the bottom plate 31 of the in-situ crystallization plate can be assembled, and the height of the plated film plate 20 is the same as the height of the side holes 32 of the in-situ crystallization plate 30, so as to facilitate the mass placement of the in-situ upper template 10 into the in-situ crystallization plate 30 and the mass removal of the in-situ upper template 10 from the in-situ crystallization plate 30 for the mass plating of the plated film plate 20.
The crystal high-throughput culture and rapid loading device provided by the preferred embodiment comprises the following use methods:
first, a plurality of in-situ upper templates 10 are placed in the first plating grooves 21 of the plating plate 20, the in-situ upper templates 10 are sealed with a film having low background scattering property, such as a polyimide film, that is, are laid flat on the upper surface of the in-situ upper templates 10, and then cut with first scores 22 arranged along the circumferential direction by a blade, so that the single-sided plating in-situ upper templates 10 can be obtained.
After the single-sided coating is completed, the uncoated side of the in-situ upper template 10 is facing upwards and is also placed in the first coating groove 21, then the coated plate chassis 25 is assembled with the in-situ crystallization plate chassis 31, as shown in fig. 6, the in-situ upper template 10 in the first coating groove 21 is sequentially pushed into the side holes 32 of the in-situ crystallization plate 30, the baffle 40 is inserted into the top groove 33 of the in-situ crystallization plate 30, the steps are repeated to form a new single-sided coating of the in-situ upper template 10, then the new single-sided coating is pushed into the side holes 32 of the in-situ crystallization plate until the in-situ upper template 10 is pushed to the baffle 13, and then the process is repeated on the other side of the in-situ crystallization plate 30 until 48 in-situ upper templates are assembled with the in-situ crystallization plate. At this time, every two spot sample holes 11 on the in-situ upper sample plate 10 and one pool liquid hole 34 of the in-situ crystallization plate 30 constitute one crystallization chamber, and each in-situ upper sample plate 10 corresponds to two pool liquid holes 34 respectively, thereby forming two crystallization chambers.
Then mother liquor is added into 96 pool liquor holes 34 of the in-situ crystallization plate 30, namely an automatic sample application machine is used for carrying out automatic sample application on the in-situ crystallization plate 30, the automatic sample application machine automatically adds the mother liquor and the protein liquor into the sample application holes 11 of the in-situ upper sample plate 10 assembled with the in-situ crystallization plate 30 according to a set proportion, and after the automatic sample application is finished, the top 35 of the in-situ crystallization plate can be sealed by using transparent adhesive tape, so that 96 closed crystallization chambers are formed.
After the protein is crystallized in the closed crystallization chamber, firstly, lightly cutting the edge of the groove 33 on the top of the in-situ crystallization plate by a knife, taking out the baffle 40, then assembling the bottom plate 25 of the coated plate with the bottom plate 31 of the in-situ crystallization plate, pushing the through plate 50 into the side hole 32 of the in-situ crystallization plate 30, namely pushing a row of 4 in-situ upper templates 10 into the second coating groove 23 of the coated plate 20 in batch, repeating the steps for 12 times until the second coating groove 23 is fully paved, sealing the in-situ upper template 10 by using a film with low background scattering property, such as a polyimide film, then cutting the adjacent in-situ upper templates 10 along the second nicks 24 on the coated plate 20 by the knife, and lightly cutting the adjacent in-situ upper templates 10 to obtain 48 in-situ upper templates 10 with double-sided coating.
After the in-situ upper template 10 with the double-sided coating is manufactured, the in-situ upper template 10 is screwed and fixed on a magnetic base (not shown) through the small head end 12 of the in-situ upper template 10, then the in-situ upper template is placed into a crystal sample box uni-puck, automatic sample loading can be carried out at a synchrotron radiation beam line station, and finally in-situ diffraction data acquisition is carried out.
It should be understood that the in-situ upper template 10, the coating plate 20 and the in-situ crystallization plate 30 provided by the present invention are not limited to the structures shown in the figures, and the number of the spotting holes, the coating grooves and the bath holes may be other numbers and any other suitable arrangements, so long as the technical effects of the present invention can be achieved, and the present invention is within the protection scope of the present invention.
The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent application. The invention has not been described in detail in order to avoid obscuring the invention.
Claims (10)
1. An in-situ based device for high-throughput culture and rapid sample loading of crystals, which is characterized by comprising:
the in-situ upper sample plates are provided with hollow sample application holes;
the coating plate is used for providing a coating place and is provided with a plurality of first coating grooves and a plurality of second coating grooves, each first coating groove has a size equivalent to that of the in-situ sample loading plate, and each second coating groove has a length which is several times that of the in-situ sample loading plate and an equal width and is used for accommodating a plurality of in-situ sample loading plates simultaneously;
the in-situ crystallization plate is used for crystal culture and comprises a plurality of pool liquid holes, the side surface of the in-situ crystallization plate also comprises a plurality of side holes which penetrate through the side surface of the in-situ crystallization plate from front to back, and the side holes are positioned above the pool liquid holes; and
the size of the through plate is matched with that of the side hole on the in-situ crystallization plate;
when the plated film plate and the in-situ crystallization plate are placed side by side on the same horizontal plane, a first plated film groove or a second plated film groove on the plated film plate is aligned with a side hole on the in-situ crystallization plate, the in-situ sample loading plate placed in the first plated film groove can enter the in-situ crystallization plate through the side hole, at least one point sample hole on the in-situ sample loading plate and one pool liquid hole of the in-situ crystallization plate form a crystallization chamber, and therefore high-throughput crystal culture and rapid sample loading based on the in-situ are achieved.
2. The apparatus of claim 1, wherein the in-situ crystallization plate has a matrix of wells, and the number of wells is x y.
3. The apparatus of claim 1, further comprising a detachable baffle plate, wherein the upper middle position of the in-situ crystallization plate along the width direction is provided with a groove extending along the whole length direction, the size of the baffle plate is matched with the groove, and the baffle plate and the adjacent crystallization chamber can form a closed gas phase diffusion space after assembly.
4. The apparatus of claim 1, wherein the in-situ crystallization plate is adapted to an automated printer.
5. The apparatus of claim 1, wherein the small end of the home upper template has a guide bevel to facilitate access to the side hole.
6. The apparatus of claim 1, wherein the coated plate and the in-situ crystallization plate can be spliced on the same horizontal plane, and the side surface of the coated plate and the side surface of the in-situ crystallization plate have a structure with a complementary shape.
7. The apparatus of claim 1, wherein the first and second plating grooves have first and second scores arranged along at least a portion of a circumferential direction, respectively, so as to facilitate cutting when plating.
8. An in-situ based crystal high-throughput culture and rapid loading method is characterized by comprising the following steps:
s1: providing an in situ based crystal high throughput culture and rapid loading device according to any one of claims 1 to 7;
s2: placing the in-situ sample loading plate in a first film coating groove on the film coating plate for back film coating, and pushing the turned in-situ sample loading plate into the in-situ crystallization plate through the side hole;
s3: carrying out sample application operation on the in-situ crystallization plate by using an automatic sample application machine, and then sealing the whole in-situ crystallization plate to form a plurality of closed crystallization chambers for carrying out protein crystallization;
s4: pushing the in-situ upper sample plates out of the in-situ crystallization plate to second coating grooves on the coating plate in batches by using a through plate, wherein each second coating groove contains a plurality of in-situ upper sample plates simultaneously, and coating the front surface of each second coating groove;
s5: and finally, fixing the in-situ upper template with the double-sided coated film on a magnetic base, and placing the template into a crystal sample box, so that automatic sample loading can be performed at a synchrotron radiation beam line station.
9. The method according to claim 8, wherein step S2 includes: sealing the in-situ sample loading plate by using a film with low background scattering property, and then cutting along a first nick on the first film coating groove by using a blade to obtain an in-situ sample loading plate with a single-sided film coating; step S4 further includes: and sealing the in-situ sample loading plate by using a film with low background scattering property, then cutting along a second nick on the second film coating groove by using a blade, and separating the adjacent in-situ sample loading plates to obtain the in-situ sample loading plate with double-sided coating.
10. The method according to claim 8, wherein step S2 further comprises: and a detachable baffle is provided, a groove extending along the whole length direction is formed in the middle position above the in-situ crystallization plate, the baffle is inserted into the groove, and the in-situ upper sample plate with a single-side coated film can be pushed into the in-situ crystallization plate from two sides through the side hole, so that the in-situ upper sample plate and the in-situ crystallization plate can be accurately assembled.
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