A serial crystallography experimental device based on conveyor belt technology

Abstract

The present invention discloses a serial crystallography experimental device based on conveyor belt technology. The device comprises a goniometer head, a base film, and a crystallization plate storage tray. The bottom end of the goniometer head is provided with a goniometer head groove for coupling with the goniometer head base on a synchrotron beamline station. The top end of the goniometer head is provided with two goniometer head support rods for coupling with bearing short rods, and a goniometer head waste liquid tank for collecting waste liquid after X-ray irradiation. The base film is coupled to the crystallization plate storage tray for supporting and flipping the crystallization plate storage tray. During the experiment, the crystallization plate storage tray, equipped with a sitting drop in situ crystallization plate, is coupled to the base film. The low background scattering film on the back of the sitting drop in situ crystallization plate is connected to the bearing short rod via an adhesive film to form a conveyor belt film. Driven by a motor, the bearing short rod tears off the conveyor belt film during sample delivery to the goniometer head and performs diffraction data acquisition. Waste sample droplets are squeezed out and flow down the goniometer head support rods into the waste liquid tank.

Description

Technical Field

[0001] The present invention relates to the field of protein crystal loading, and more particularly to an experimental device and method based on a conveyor belt loading technology. Background Art

[0002] As a primary technique for protein structure elucidation, crystallography has seen a gradual shift in focus in recent years towards serial crystallography, which has become a new research priority due to its potential for high-throughput loading and time resolution. Serial loading technology, a key component of serial crystallography, has seen rapid development, encompassing fixed target technologies such as chips, in situ plates, and nylon loops, as well as mobile phase techniques such as high-viscosity nozzles, electrospinning, and capillaries.

[0003] In serial crystallography, membrane-based in situ plate technology has been widely studied due to its high-throughput potential. Membrane-based in situ plate technology is not only compatible with existing high-throughput automated platforms, but also avoids the tedious work of crystal retrieval by using an in situ method. Existing membrane-based in situ plate technology mainly performs fixed target loading by cutting the in situ small plate from the in situ plate, but this method limits the loading efficiency of the sample. At the same time, this loading method also limits the use scenarios of time-resolved technology, and conventional mobile phase time-resolved technology cannot be applied. Existing serial crystallography based on conveyor belt technology also has some limitations. The first is how to efficiently culture protein crystals, and the second is how conveyor belt-based technology can efficiently transfer crystals to a conveyor belt with low background scattering. Summary of the Invention

[0004] In order to solve the problems of efficiency of existing membrane-based in-situ plate technology and conveyor belt technology, the present invention provides a conveyor belt-based serial crystallography experimental device and method, which integrates the advantages of the two technologies, improves the in-situ sampling efficiency and time resolution potential, and at the same time improves the sample preparation efficiency of the conveyor belt method itself and the efficiency of sample transmission to the conveyor belt.

[0005] According to the conveyor belt loading device of the present invention, it mainly includes an angle measuring head end 10, a bottom film end 20, a crystallization plate storage tray 30, a sitting drop in situ crystallization plate 40, a short bearing rod 50, a long bearing rod 60, and a bearing rod cap 70.

[0006] The crystallization plate storage disk 30 can be assembled on the bottom film end 20 through a bearing long rod 60. One end of the bearing long rod 60 is provided with a long rod thread 62 which can pass through the two storage plate bearing holes 31 and the two bottom film end bearing holes 21. The bearing rod cap 70 completes the fixation of the crystallization plate storage disk 30 and the bottom film end 20 by assembling the rod cap thread 71 on its inner wall with the long rod thread 62.

[0007] The sitting drop in-situ crystallization plate 40 is assembled with the crystallization plate storage tray 30 through the storage tray groove 32 on the crystallization plate storage tray 30 . The crystallization plate storage tray 30 can be assembled with one or more sitting drop in-situ crystallization plates.

[0008] One end of the long bearing rod 60 features long rod threads 62, which can be passed through the two bottom membrane end bearing holes 23. The bearing rod cap 70, with its inner wall threaded with rod cap threads 71, mates with the long rod threads 62. Similarly, one end of the short bearing rod 50 features short rod threads 52, which can be passed through the two goniometer end bearing holes 13 on the goniometer head 10. The bearing rod cap 70, again with rod cap threads 71, mates with the short rod threads 52. The goniometer head 10 has two functions: first, it mates with the goniometer head, facilitating adjustment of the conveyor belt and sample droplet positions using the goniometer head base; second, it serves as the fixed end of the conveyor belt, working in conjunction with the bottom membrane end to form the entire conveyor belt assembly.

[0009] The goniometer head end 10 is assembled with the goniometer head base on the synchrotron radiation beamline station through the goniometer head groove 14 .

[0010] The goniometer head end 10 can be connected to the low-background scattering film of the sitting-drop in-situ crystallization plate 40 on the base film end 20 through an adhesive film to form a conveyor belt film. The crystallization plate storage disk 30 is rotated 180 degrees clockwise, and the motor drives the short rod head 51 of the goniometer head end 10 to continuously transport the sample on the sitting-drop in-situ crystallization plate 40 to the goniometer head end 10, and diffraction data is collected during this process.

[0011] The conveyor belt film is continuously wound onto the bearing short rod 50 , and the discarded sample droplets are squeezed out and flow down along the goniometer head end support rod 12 and finally flow into the goniometer head end waste liquid tank 11 .

[0012] The crystallization plate storage tray 30 is rotated 180° counterclockwise to complete the reset, and the conveyor belt film is reconnected with the low background scattering film of the sitting drop in-situ crystallization plate 40 to form a new conveyor belt film.

[0013] Preferably, the goniometer head end 10 is provided with two goniometer head end support rods 12, on which there are two goniometer head end through-membrane bearing holes 13 for assembling the bearing short rods 50. The device is also provided with a goniometer head end waste liquid tank 11 and a goniometer head groove 14 for collecting waste liquid after X-ray irradiation and fixing the goniometer head base, respectively.

[0014] Preferably, the bottom film end 20 is provided with two bottom film end bearing holes 21 and two bottom film end through-film bearing holes 23 for assembly with the bearing long rod 60. The device is also provided with a bottom film end protrusion 22 for correcting and supporting the crystallization plate storage tray 30.

[0015] Preferably, the crystallization plate storage tray 30 is provided with two storage tray bearing holes 31 for assembly with the bearing rod 60. The two storage tray bearing holes 31 are located at a non-center upper position on the side of the crystallization plate storage tray 30 so that when the crystallization plate storage tray 30 is rotated 180 degrees clockwise, the crystallization plate storage tray 30 is flipped over and supported by the base film end protrusion 22. The device also has a storage tray groove 32 for assembly with the sitting drop in-situ crystallization plate 40.

[0016] Preferably, the bearing short rod 50 is provided with a short rod head 51 and a short rod thread 52. An external motor can be assembled with the short rod head 51 of the bearing short rod 50 to drive the bearing short rod 50 to rotate. The short rod thread 52 is assembled with the bearing rod cap 70 through the rod cap thread 71.

[0017] Preferably, the bearing long rod 60 is provided with a long rod head 61 and a long rod thread 62. The long rod thread 62 is assembled with the bearing rod cap 70 through a rod cap thread 71.

[0018] The present invention also provides a protein crystal loading method, which comprises the following steps:

[0019] 1) Seal the front side of each sitting drop in situ crystallization plate 40 with transparent tape, and seal the back side of each sitting drop in situ crystallization plate 40 with several strips of low-background scattering film, with a certain length left at both ends of each low-background scattering film as film strip heads;

[0020] 2) Assembling a plurality of the sitting drop in-situ crystallization plates 40 with their reverse sides facing upward onto the crystallization plate storage tray 30;

[0021] 3) connecting the film strips of the low background scattering film at the same position on each of the sitting drop in situ crystallization plates 40 to each other and then connecting them to an adhesive film to form a conveyor belt film;

[0022] 4) The i-th conveyor belt film is connected to the bearing short rod 50, and the crystallization plate storage disk 30 is rotated 180 degrees clockwise. The bearing short rod 50 is driven by a motor to rotate clockwise. During the process of continuously transporting the sample sealed by the i-th conveyor belt film on each sitting drop in situ crystallization plate 40 to the goniometer head 10, the i-th conveyor belt film is gradually torn off. X-rays are passed through the low background scattering film and irradiated on the sample to collect diffraction data. After sample collection is completed, the conveyor belt film is continuously rolled onto the bearing short rod 50, and the discarded sample droplets are squeezed out and flow down along the goniometer head support rod 12 into the goniometer head waste liquid tank 11.

[0023] 5) Rotate the crystallization plate storage disk 30 180° counterclockwise, connect the (i+1)th conveyor belt film to the (i)th conveyor belt film via the membrane head, and continue to collect samples.

[0024] Compared with the prior art, the present invention has the following positive effects:

[0025] This device and method combine the advantages of both membrane-based in-situ plate technology and conveyor belt technology: The membrane-based in-situ plate technology is compatible with existing high-throughput automated platforms, enabling efficient cultivation of protein crystals. Its in-situ approach eliminates the tedious task of extracting crystals. The conveyor belt technology efficiently transfers crystals into the X-ray path and has the potential to enable mobile phase time-resolved techniques. This method creatively addresses the challenge of rapidly splicing low-background scattering membranes from membrane-based in-situ plates to form a conveyor belt. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of an experimental device for conveyor belt loading technology according to a preferred embodiment of the present invention.

[0027] Figure 2 2 is a schematic structural diagram of a goniometer head according to a preferred embodiment of the present invention.

[0028] Figure 3 2 is a schematic diagram of the bottom structure of a goniometer head according to a preferred embodiment of the present invention.

[0029] Figure 4 Schematic diagram of the structure of the bottom film end according to a preferred embodiment of the present invention.

[0030] Figure 5 It is a schematic structural diagram of a crystallization plate storage disk according to a preferred embodiment of the present invention.

[0031] Figure 6 It is a schematic structural diagram of a sitting drop in-situ crystallization plate according to a preferred embodiment of the present invention.

[0032] Figure 7 It is a schematic diagram of the bottom structure of a sitting drop in-situ crystallization plate according to a preferred embodiment of the present invention.

[0033] Figure 8 It is a structural schematic diagram of a bearing short rod according to a preferred embodiment of the present invention.

[0034] Figure 9 It is a structural schematic diagram of a bearing long rod according to a preferred embodiment of the present invention.

[0035] Figure 10 1 is a schematic structural diagram of a bearing rod cap according to a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are given below in conjunction with the accompanying drawings and described in detail.

[0037] Example

[0038] like Figure 1As shown, the serial crystallography experimental device based on the conveyor belt technology mainly includes a goniometer head end 10, a base film end 20, a crystallization plate storage tray 30, a sitting drop in situ crystallization plate 40, a short bearing rod 50, a long bearing rod 60, and a bearing rod cap 70.

[0039] like Figure 2 、 Figure 3 As shown, the bottom end of the goniometer head 10 is provided with a goniometer head groove 14 for assembly with the goniometer head base on the synchrotron radiation beamline station; the top end of the goniometer head 10 is provided with two goniometer head support rods 12 for assembly with the bearing short rod 50; the top end of the goniometer head 10 is provided with a goniometer head waste liquid tank 11 for collecting waste liquid after X-ray irradiation; the top end of the goniometer head 10 is a groove, the center of which is provided with a cylinder, and the cylinder is provided with two goniometer head support rods 12; the goniometer head waste liquid tank 11 is formed between the cylinder and the side wall of the groove.

[0040] like Figure 4 As shown, the bottom film end 20 includes two supporting plates and a rear end plate connecting the two supporting plates; the crystallization plate storage disk 30 is assembled on the bottom film end 20 through a first bearing long rod and a bearing rod cap; wherein, storage disk bearing holes 31 are respectively provided on the two side plates of the crystallization plate storage disk 30, and bottom film end bearing holes 21 are respectively provided on the two supporting plates of the bottom film end 20, and a long rod thread is provided at one end of the first bearing long rod, and the long rod thread passes through the two storage disk bearing holes 31, the two bottom film end bearing holes 21 and the rod cap thread on the inner wall of the bearing rod cap to complete the assembly of the bottom film end 20 and the crystallization plate storage disk 30.

[0041] like Figure 5 As shown, the crystallization plate storage tray 30 includes two side plates and a front end plate connecting the two side plates, the bottom ends of the side plates are provided with inwardly protruding edges, the bottom end of the front end plate is provided with an insertion port, the insertion port and the protruding edge constitute a storage tray groove 32, the sitting drop in situ crystallization plate 40 is inserted into the crystallization plate storage tray 30 through the storage tray groove 32, thereby realizing the assembly of the sitting drop in situ crystallization plate 40 and the crystallization plate storage tray 30.

[0042] When conducting crystallographic experiments, a conventional sitting drop in situ crystallization plate 40 is used. The sitting drop in situ crystallization plate 40 has 96 crystallization chambers 41 arranged in 12 rows and 8 columns. Each crystallization chamber has a mother liquor tank 43 and a crystallization hole 42. Figure 6 As shown, the front side of the sitting drop in situ crystallization plate 40 is sealed with a transparent tape, as shown in FIG. Figure 7As shown, the back side of the sitting drop in situ crystallization plate 40 is sealed with a low-background scattering film. Unlike the front side of the sitting drop in situ crystallization plate which is sealed with a whole piece of transparent tape, this experiment uses four strip-shaped low-background scattering films to seal the back side of the sitting drop in situ crystallization plate 40. Each strip-shaped low-background scattering film can seal two columns of crystallization holes 42 on the back side of the sitting drop in situ crystallization plate. Four strip-shaped low-background scattering films can seal eight columns of crystallization holes on the back side of the sitting drop in situ crystallization plate. A certain amount of excess length of film is left at both ends of the four strip-shaped low-background scattering films, which is called a membrane head.

[0043] After the sitting drop in situ crystallization plate 40 has finished culturing the sample, its reverse side faces upward and can be assembled with the crystallization plate storage plate 30 through the storage plate groove 32, as shown in FIG. Figure 1 As shown, the reverse side of the sitting drop in situ crystallization plate 40 is assembled on the crystallization plate storage tray 30 with the adhesive low background scattering film facing upward. The crystallization plate storage tray 30 can be assembled with one or more sitting drop in situ crystallization plates 40. In this experiment, we take the crystallization plate storage tray device that can store two sitting drop in situ crystallization plates 40 as an example to describe the entire experimental method. After the two sitting drop in situ crystallization plates 40 are assembled on the crystallization plate storage tray 30, the crystallization plate storage tray 30 is placed as shown in the following figure. Figure 1 The crystallization plate storage tray 30 is assembled with the bottom film end 20 in the manner shown. The right end of the crystallization plate storage tray 30 now rests against and to the left of the bottom film end protrusion 22. Align the two storage tray bearing holes 31 with the two bottom film end bearing holes 21. Insert the long rod thread 62 of the bearing rod 60 through the two storage tray bearing holes 31 and the two bottom film end bearing holes 21. The bearing rod cap 70, through the cap thread 71, engages the long rod thread 62 to secure the crystallization plate storage tray 30 to the bottom film end 20.

[0044] The long rod thread 62 of the bearing long rod 60 is then passed through the two bottom membrane end through-membrane bearing holes 23. The bearing rod cap 70 is again assembled with the long rod thread 62 through the rod cap thread 71 on its inner wall. Similarly, the short rod thread 52 of the bearing short rod 50 is passed through the two goniometer end through-membrane bearing holes 13. The bearing rod cap 70 is again assembled with the short rod thread 52 through the rod cap thread 71. Finally, the goniometer head end 10 is assembled with the goniometer head base through the goniometer head groove 14.

[0045] After the device is assembled, select a piece of adhesive film with the same width as the strip of low background scattering film, such as Figure 1As shown, the sitting drop in situ crystallization plate at the left end of the crystallization plate storage tray 30 is used as plate No. 1, and the sitting drop in situ crystallization plate at the right end of the crystallization plate storage tray 30 is used as plate No. 2. The sticky end of the sticky film is downward and connected to any one of the four film heads at the right end of plate No. 2, and the film head at the left end of plate No. 2 can also be connected to the corresponding film head at the right end of plate No. 1. At this time, the sticky film is connected to the low background scattering film on plates No. 1 and 2 to form a conveyor belt film. The conveyor belt film passes through the bottom film end 20, that is, it is located under the two bearing long rods 60 on the bottom film end 20. The conveyor belt film has the sticky end downward, and its right end passes over the top of the bearing short rod 50 on the goniometer head end 10 and adheres to the bearing short rod 50, clockwise 180 °Rotate the crystallization plate storage tray 30, and after the crystallization plate storage tray 30 is flipped over, it is supported by the protrusion 22 at the bottom film end. At this time, the positions of plate No. 1 and plate No. 2 are interchanged, and the external motor can be assembled with the short rod head 51 of the bearing short rod 50. Drive the external motor to make the short rod head 51 rotate clockwise, and the conveyor belt film on plate No. 2 and plate No. 1 is torn off. The device can continuously transport the sample on the sitting drop in situ crystallization plate to the goniometer head end 10. During this process, X-rays can pass through the low background scattering film and irradiate the sample to collect diffraction data. After the sample collection is completed, the conveyor belt film is continuously rolled on the bearing short rod 50, and the discarded sample droplets are squeezed out and flow down along the goniometer head end support rod 12 and finally merge into the goniometer head end waste liquid tank 11.

[0046] When the transmission of samples on one conveyor belt film is completed, that is, the transmission of two rows of samples on plates No. 1 and No. 2 is completed, the crystallization plate storage disk 30 is rotated 180° counterclockwise to reset it, and the conveyor belt film is reconnected to the other remaining film heads on the right end of plate No. 2. Repeat the operation to form a new conveyor belt film and finally transmit all samples on the sitting drop in situ crystallization plate 40 to the X-ray path.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Various modifications are possible. In other words, any simple, equivalent changes and modifications made in accordance with the claims and description of the present invention are within the scope of protection of the patent claims. Anything not fully described in this invention constitutes conventional technical content.

Claims

1. A serial crystallography experimental device based on conveyor belt technology, characterized in that: It includes an angle measuring head end (10), a bottom film end (20), and a crystallization plate storage tray (30); The bottom end of the goniometer head end (10) is provided with a goniometer head groove (14) for assembling with a goniometer head base on a synchrotron radiation beamline station; the top end of the goniometer head end (10) is provided with two goniometer head end support rods (12) for assembling with a bearing short rod (50); the top end of the goniometer head end (10) is provided with a goniometer head end waste liquid tank (11) for collecting waste liquid after being irradiated by X-rays; The crystallization plate storage tray (30) is used to store the sitting drop in-situ crystallization plate (40); The bottom film end (20) is assembled with the crystallization plate storage tray (30) and is used to support and flip the crystallization plate storage tray (30); During the experiment, the crystallization plate storage tray (30) equipped with the sitting drop in situ crystallization plate (40) is assembled onto the bottom film end (20), and the low background scattering film on the back side of the sitting drop in situ crystallization plate (40) is connected to the bearing short rod (50) through an adhesive film to form a conveyor belt film. The bearing short rod (50) is driven by a motor to transport the sample on the sitting drop in situ crystallization plate (40) to the goniometer head end (10), tearing off the conveyor belt film and collecting diffraction data during the process, and the discarded sample droplets are squeezed out and flow down along the goniometer head end support rod (12) into the goniometer head end waste liquid tank (11).

2. The serial crystallography experimental device according to claim 1, characterized in that The crystallization plate storage tray (30) comprises two side plates and a front end plate connecting the two side plates, the bottom ends of the side plates are provided with inwardly protruding edges, the bottom end of the front end plate is provided with an insertion port, the insertion port and the protruding edge form a storage tray groove (32), and the sitting drop in-situ crystallization plate (40) is inserted into the crystallization plate storage tray (30) through the storage tray groove (32), thereby realizing the assembly of the sitting drop in-situ crystallization plate (40) and the crystallization plate storage tray (30).

3. The serial crystallography experimental device according to claim 2, characterized in that The bottom film end (20) includes two supporting plates and a rear end plate connecting the two supporting plates; the crystallization plate storage disk (30) is assembled on the bottom film end (20) through a first bearing long rod and a bearing rod cap; wherein, storage disk bearing holes (31) are respectively provided on the two side plates of the crystallization plate storage disk (30), and bottom film end bearing holes (21) are respectively provided on the two supporting plates of the bottom film end (20); one end of the first bearing long rod is provided with a long rod thread, and the long rod thread passes through the two storage disk bearing holes (31), the two bottom film end bearing holes (21) and the rod cap thread on the inner wall of the bearing rod cap, thereby completing the assembly of the bottom film end (20) and the crystallization plate storage disk (30).

4. The serial crystallography experimental device according to claim 3, characterized in that A bottom film end protrusion (22) is provided at the bottom end of the rear end plate of the bottom film end (20) for correcting and supporting the crystallization plate storage tray (30).

5. The serial crystallography experimental device according to claim 4, characterized in that: The two storage plate bearing holes (31) are located at a non-center upper position on the side of the crystallization plate storage plate (30) so that when the crystallization plate storage plate (30) is rotated 180 degrees clockwise, the crystallization plate storage plate (30) is turned over and supported by the bottom film end protrusion (22).

6. The serial crystallography experimental device according to claim 3, characterized in that Two supporting rods are provided on the rear end plate of the bottom film end (20), and each supporting rod is provided with a bottom film end through-film bearing hole (23) for assembling with the second bearing long rod; the adhesive film is connected to the bearing short rod (50) via the second bearing long rod to form a conveyor belt film.

7. The serial crystallography experimental device according to any one of claims 1 to 6, characterized in that: Each goniometer head end support rod (12) is provided with a goniometer head end through-membrane bearing hole (13), and one end of the bearing short rod (50) is provided with a short rod thread (52); the short rod thread (52) passes through the two goniometer head end through-membrane bearing holes (13) and is connected to the rod cap thread (71) on the inner wall of the bearing rod cap (70), thereby completing the assembly of the goniometer head end support rod (12) and the bearing short rod (50).

8. The serial crystallography experimental device according to claim 7, characterized in that: The top end of the goniometer head (10) is a groove, a cylinder is provided at the center of the groove, and two goniometer head support rods (12) are provided on the cylinder; The goniometer head end waste liquid tank (11) is formed between the cylinder and the side wall of the groove.

9. The serial crystallography experimental device according to claim 1, characterized in that: The crystallization plate storage tray (30) stores a plurality of side-by-side sitting drop in-situ crystallization plates (40); a plurality of rows of crystallization holes (42) on the reverse side of each sitting drop in-situ crystallization plate are sealed by a low-background scattering film, and the film strips of the low-background scattering films at corresponding positions on adjacent sitting drop in-situ crystallization plates (40) are connected together and then connected to the bearing short rod (50) through an adhesive film to form a conveyor belt film.

10. A method for loading protein crystals based on the serial crystallography experimental apparatus of claim 1, comprising: 1) using a transparent tape to seal the front side of each sitting drop in situ crystallization plate (40), and using a plurality of low-background scattering films to seal the back side of each sitting drop in situ crystallization plate (40), with a certain length left at both ends of each low-background scattering film as a film tape head; 2) assembling a plurality of the sitting drop in-situ crystallization plates (40) with their reverse sides facing upward onto the crystallization plate storage tray (30); 3) connecting the film strips of the low background scattering film at the same position on each of the sitting drop in situ crystallization plates (40) to each other and then connecting them with an adhesive film to form a conveyor belt film; 4) Connecting the i-th conveyor belt film to the bearing short rod (50), rotating the crystallization plate storage disk (30) 180 degrees clockwise, and using a motor to drive the bearing short rod (50) to rotate clockwise, the i-th conveyor belt film is gradually torn off during the process of continuously transporting the sample sealed by the i-th conveyor belt film on each sitting drop in-situ crystallization plate (40) to the goniometer head end (10), and using X-rays to pass through the low background scattering film and irradiate the sample to collect diffraction data. After the sample collection is completed, the conveyor belt film is continuously rolled on the bearing short rod (50), and the discarded sample droplets are squeezed out and flow down along the goniometer head end support rod (12) and merged into the goniometer head end waste liquid tank (11); 5) Rotate the crystallization plate storage disk (30) 180° counterclockwise, connect the (i+1)th conveyor belt film to the (i)th conveyor belt film through the membrane head, and continue to collect samples.

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