Preparation kit of sample for low-temperature electron microscope and application of preparation kit

Abstract

A kit for preparing a sample for a low-temperature electron microscope is provided with an amphiphilic protein and a protein cross-linking agent, or contains an expression vector that encodes an amphiphilic nucleic acid. A kit for preparing a sample for a low-temperature electron microscope is provided with an ice thickness control molecule to which an amphiphilic protein is bound. The amphiphilic protein may have an amphiphilic alpha helical structure. The amphiphilic protein can be anti-freezing protein and can also be I-type anti-freezing protein.

Description

Technical Field

[0001] The present invention relates to a kit for preparing a specimen for cryo-electron microscopy and its applications. Specifically, the present invention relates to a kit for preparing a specimen for cryo-electron microscopy, a method for preparing a specimen for cryo-electron microscopy, a method for controlling the orientation of a target protein in a specimen for cryo-electron microscopy, a method for suppressing the collision and adsorption of a target protein in a specimen for cryo-electron microscopy to an amorphous carbon film, and a method for controlling the thickness of ice in a specimen for cryo-electron microscopy. This application claims priority based on Japanese Patent Application No. 2022-204279 filed in Japan on December 21, 2022, and incorporates its content herein. Background Art

[0002] Conventionally, as methods for analyzing the structures of biomolecules such as proteins, X-ray crystallography, NMR methods, etc. have been used. However, in recent years, single particle analysis using cryo-electron microscopy (hereinafter simply referred to as "cryo-electron microscopy method") has attracted attention. In the cryo-electron microscopy method, crystallization of the specimen, which is necessary for X-ray crystal structure analysis, is not required, and there is no upper limit on the molecular weight of the specimen that restricts the application of the NMR method.

[0003] However, in biomolecules, most of the interatomic bonds that maintain their three-dimensional structures are non-covalent bonds such as hydrogen bonds and ionic bonds. Therefore, they are more susceptible to electron beam irradiation damage compared to specimens such as metals and semiconductors. Thus, in the cryo-electron microscopy method, in order to record projection images (hereinafter referred to as "particle images") of biomolecule particles with high resolution with high image quality while suppressing damage as much as possible, it is required to rapidly freeze an aqueous solution of biomolecules to embed the biomolecules in an amorphous ice film as a thin film, irradiate an electron beam while maintaining a low temperature constantly on a specimen stage cooled with liquid helium or liquid nitrogen, and record an electron microscope image. In addition, depending on the type of biomolecule, sometimes the biomolecule adsorbs to the amorphous carbon film of the specimen grid, and the biomolecules cannot be dispersed throughout the holes in which the amorphous ice film is formed as the observation area.

[0004] Furthermore, each particle image is a projection image of a molecule embedded in an ice film in various orientations. Therefore, in order to reconstruct a three-dimensional image, it is also necessary to collect a large number of images projected in various directions as uniformly as possible. That is, it is necessary to efficiently collect as many particle images as possible, classify and average them for each projection direction on the basis of making the orientations and positions of the particle images consistent, thereby reducing the noise level and increasing the signal level. And by determining the relative relationships of the projection directions of each particle image and performing back-projection, a high-resolution three-dimensional image can be reconstructed for the first time. However, sometimes only projection images of particles that are partially damaged due to collision with the gas-liquid interface or projection images of particles taken from a specific direction due to adsorption to the gas-liquid interface are obtained.

[0005] On the other hand, antifreeze proteins, known as amphiphilic proteins, are relatively small proteins composed of polypeptides of about 30 or more and 150 or fewer amino acid residues. In a temperature region below 0°C, they bind to specific crystal planes of ice nuclei and inhibit their growth, thereby having the ability to prevent the freezing of water or water-containing substances. In recent years, it has been clarified that antifreeze proteins have the property of aggregating at the air-liquid interface (for example, refer to Non-Patent Document 1, etc.). Prior Art Documents Non-Patent Documents

[0006] Non-Patent Document 1: Meister K et al., “Investigation of the Ice-Binding Site of an Insect Antifreeze Protein Using Sum-Frequency Generation Spectroscopy.”, J. Phys. Chem. Lett., Vol. 6, Issue 7, pp. 1162 - 1167, 2015. Summary of the Invention Problems to be Solved by the Invention

[0007] The present invention has been completed in view of the above circumstances, and provides a preparation kit for a specimen for a cryo-electron microscope, which can suppress the deviation of the posture of a target protein in a specimen from an undesired direction with respect to the electron beam incident direction, as well as the collision and adsorption to the air-liquid interface, and can obtain a specimen for a cryo-electron microscope in which the target protein is dispersed throughout the observation region. In addition, a preparation kit for a specimen for a cryo-electron microscope capable of uniformly controlling the thickness of ice in the specimen is provided. Furthermore, a method for preparing a specimen for a cryo-electron microscope using the above-mentioned preparation kit for a specimen for a cryo-electron microscope, a method for controlling the posture of a target protein in a specimen for a cryo-electron microscope, a method for suppressing the adsorption of a target protein in a specimen for a cryo-electron microscope to an amorphous carbon film, a method for suppressing the collision and adsorption of a target protein in a specimen for a cryo-electron microscope to the air-liquid interface, and a method for controlling the thickness of ice in a specimen for a cryo-electron microscope are provided. Means for Solving the Problems

[0008] That is, the present invention includes the following aspects. (1) A preparation kit for a specimen for a cryo-electron microscope, which comprises an amphiphilic protein or an expression vector containing a nucleic acid encoding the amphiphilic protein. (2) A preparation kit for a specimen for a cryo-electron microscope, which comprises an ice thickness control molecule to which an amphiphilic protein is bound. (3) The kit for preparing a specimen for cryo-electron microscopy according to (1) or (2), wherein the amphiphilic protein has an amphiphilic α-helical structure. (4) The kit for preparing a specimen for cryo-electron microscopy according to any one of (1) to (3), wherein the amphiphilic protein is an antifreeze protein. (5) The kit for preparing a specimen for cryo-electron microscopy according to (4), wherein the antifreeze protein is a type I antifreeze protein. (6) The kit for preparing a specimen for cryo-electron microscopy according to (5), wherein the type I antifreeze protein is HPLC6 peptide. (7) The kit for preparing a specimen for cryo-electron microscopy according to any one of (1) to (3), wherein the amphiphilic protein is KALA peptide. (8) The kit for preparing a specimen for cryo-electron microscopy according to (1), further comprising a protein crosslinking agent. (9) The kit for preparing a specimen for cryo-electron microscopy according to (8), wherein the protein crosslinking agent is an amine-reactive crosslinking agent. (10) The kit for preparing a specimen for cryo-electron microscopy according to (9), wherein the amine-reactive crosslinking agent is glutaraldehyde, or bis(3-sulfo-N-succinimidyl) suberate or a salt thereof. (11) The kit for preparing a specimen for cryo-electron microscopy according to (1), further comprising an ice thickness control molecule conjugated with an amphiphilic protein. (12) The kit for preparing a specimen for cryo-electron microscopy according to (2) or (11), wherein the ice thickness control molecule is an antifreeze protein and is a multimeric protein. (13) The kit for preparing a specimen for cryo-electron microscopy according to (12), wherein the multimeric protein is glutamine synthetase or ferritin. (14) A method for preparing a specimen for cryo-electron microscopy, comprising: using the kit for preparing a specimen for cryo-electron microscopy according to any one of (1) to (13) to prepare a specimen for cryo-electron microscopy containing a target protein. (15) A method for controlling the conformation of a target protein in a specimen for cryo-electron microscopy, comprising: using the kit for preparing a specimen for cryo-electron microscopy according to any one of (1), (8) to (11) to prepare a specimen for cryo-electron microscopy containing a target protein. (16) A method for inhibiting the adsorption of a target protein in a specimen for a cryo-electron microscope to an amorphous carbon film, comprising: preparing a specimen for a cryo-electron microscope containing the target protein using the specimen preparation kit for a cryo-electron microscope according to any one of (1), (8) to (11). (17) A method for inhibiting the collision and adsorption of a target protein in a specimen for a cryo-electron microscope to a gas-liquid interface, comprising: preparing a specimen for a cryo-electron microscope containing the target protein using the specimen preparation kit for a cryo-electron microscope according to any one of (1), (8) to (11). (18) A method for controlling the thickness of ice in a specimen for a cryo-electron microscope, comprising: preparing a specimen for a cryo-electron microscope containing the target protein using the specimen preparation kit for a cryo-electron microscope according to any one of (2), (11) to (13). Advantages of the Invention

[0009] For the specimen for a cryo-electron microscope according to the above method, a specimen for a cryo-electron microscope can be obtained in which the deviation of the undesired posture of the target protein in the specimen with respect to the electron beam incident direction and the collision and adsorption to the gas-liquid interface are inhibited, and the target protein is dispersed throughout the observation region. In addition, for the specimen for a cryo-electron microscope according to the above method, a specimen for a cryo-electron microscope with the thickness of the ice in the specimen uniformly controlled can be obtained. Brief Description of the Drawings

[0010] Figure 1 It is a two-dimensional classification average image of a particle image of hemagglutinin protein conjugated with HPLC6 peptide taken by a cryo-electron microscope in Example 1. Figure 2A It is an image of glutamine synthetase taken by a cryo-electron microscope in Example 2. Figure 2B It is an image of glutamine synthetase conjugated with HPLC6 peptide taken by a cryo-electron microscope in Example 2. Figure 3 It is an image of ferritin and ferritin conjugated with HPLC6 peptide taken by a cryo-electron microscope in Example 3. Figure 4 It is an image of a mixed solution of hemagglutinin protein and ferritin conjugated with HPLC6 peptide taken by a cryo-electron microscope in Example 4. Figure 5 It is a two-dimensional classification average image of a particle image of Inosine-5’-monophosphatedehydrogenase2 (IMPDH2) taken by a cryo-electron microscope in Example 5. Figure 6AIt is an image of ferritin taken by cryo-electron microscopy in Example 6. Figure 6B It is an image of the fusion protein of ferritin and KALA peptide taken by cryo-electron microscopy in Example 6. Detailed implementation manners

[0011] "Preparation Kit for Specimens for Cryo-Electron Microscopy" <First Embodiment> The preparation kit for specimens for cryo-electron microscopy according to one embodiment of the present invention (hereinafter, sometimes referred to as "the preparation kit of the present embodiment") includes an antifreeze protein or an expression vector containing a nucleic acid encoding the antifreeze protein.

[0012] In cryo-electron microscopy, in order to reconstruct the three-dimensional image of the target protein, it is necessary to collect as many images as possible that project the target protein in various directions as uniformly as possible. However, in the specimen grid for cryo-electron microscopy, sometimes there is a deviation in the posture of the protein with respect to the incident direction of the electron beam, and the particle images with the same orientation account for the majority. In addition, sometimes the protein adsorbs to the amorphous carbon film that only has pores (about 1 μm in diameter) serving as the observation area of the grid, and cannot be evenly dispersed inside the observation area, and a sufficient number of particle images cannot be obtained. Furthermore, sometimes only the projection images of particles that are partially damaged due to collision with the gas-liquid interface or the projection images of particles taken from a specific direction due to adsorption to the gas-liquid interface are obtained.

[0013] In contrast, according to the property that the antifreeze protein aggregates at the gas-liquid interface, the preparation kit of the present embodiment can protect the gas-liquid interface by mixing with the antifreeze protein for the target protein that is likely to adsorb to the gas-liquid interface in a specific posture, and can inhibit the target protein from colliding with or adsorbing to the gas-liquid interface. Thereby, it is possible to reduce the particle images of the target protein that are partially damaged, and to reduce and eliminate the preferred orientation of the target protein particles. That is, it is possible to inhibit the deviation of the posture of the protein with respect to the incident direction of the electron beam and obtain the projection images of the protein in various postures.

[0014] In addition, according to the property that the above-mentioned antifreeze protein aggregates at the gas-liquid interface, the preparation kit of the present embodiment can inhibit the adsorption of the target protein to the amorphous carbon film and make the target protein evenly dispersed inside the observation area (pores) of the grid.

[0015] It should be noted that in this specification, the cryo-electron microscopy method refers to the following method: A sample solution of biomolecules such as proteins after separation and purification is rapidly frozen to embed the biomolecules in a thin ice film, and a special transmission electron microscope (TEM) that can irradiate electron rays while maintaining the sample at a low temperature under the cooling of liquid nitrogen (about -196 °C) is used to obtain several hundred to several thousand images, and the three-dimensional structure of the target biomolecule is reconstructed from the obtained electron microscope images through computer image processing.

[0016] In addition, in this specification, the sample for cryo-electron microscopy is prepared by rapidly freezing a solution containing the target protein on a holey grid. The holey grid for cryo-electron microscopy is composed of a structure in which a support film with regularly arranged holes is further pasted on a metal mesh with a lattice pattern having holes made of a conductive material such as copper, gold, or molybdenum. The support film usually uses a thin carbon film of about 10 nm or more and 50 nm or less, but in order to prevent the charging of the film caused by electron beam irradiation, a film formed by coating a gold thin film of about 5 nm or more and 10 nm or less thereon is also used.

[0017] [Antifreeze protein] Antifreeze proteins are proteins found in fish and the like that inhabit low-temperature environments. They are proteins that lower the freezing point (temperature at freezing) in organisms and help maintain the life of organisms by preventing freezing and preventing the recrystallization of ice crystals. Antifreeze proteins strongly bind to the surfaces of countless tiny ice crystals that form inside water at the moment of freezing and inhibit their growth.

[0018] Antifreeze proteins derived from fish are classified into four types according to their structures. Type I antifreeze protein is an α-helical protein containing a large number of alanine residues and having threonine residues and aspartic acid residues arranged at equal intervals. Type II antifreeze protein is a C-type lectin-like protein containing disulfide bonds. Type III antifreeze protein is a globular protein composed of characteristic structural motifs. Type IV antifreeze protein is a protein with an unknown three-dimensional structure containing a large number of glutamine residues. Among them, from the aspects of known crystal structure, small molecular weight, simple structure, and no interference with the projection of the particle image of the target protein, Type I antifreeze protein is preferred. As a preferred Type I antifreeze protein, for example, HPLC6 peptide composed of 37 amino acid residues (amino acid sequence: SEQ ID NO: 1) can be cited. The HPLC6 peptide shown in SEQ ID NO: 1 is a partial amino acid sequence of an antifreeze protein from Pseudopleuronectes americanus. The full-length amino acid sequence of the antifreeze protein from Pseudopleuronectes americanus is described, for example, in GenBank accession numbers AB59964.1, CAA30389.1, AAA49469.1, AAA49471.1, AAA49472.1, etc.

[0019] In addition, in the preparation kit of the present embodiment, when an expression vector containing a nucleic acid encoding an antifreeze protein is used, by inserting a nucleic acid encoding a target protein upstream or downstream of the nucleic acid, the antifreeze protein can be bound to any position of the target protein. Thus, due to the property of the antifreeze protein to aggregate at the gas-liquid interface, the surface of the target protein bound with the antifreeze protein faces the gas-liquid interface, thereby being able to suppress the deviation of the posture of the protein with respect to the incident direction of the electron beam and obtaining projection images of proteins in various postures.

[0020] [Protein crosslinking agent] Alternatively, the preparation kit of the present embodiment can bind the antifreeze protein to any position of the target protein by using a combination of an antifreeze protein and a protein crosslinking agent.

[0021] As the protein crosslinking agent, substances commonly used as crosslinking agents between proteins can be used. Specifically, as the protein crosslinking agent, amine-reactive crosslinking agents that crosslink primary amino groups (-NH2), thiol-reactive crosslinking agents that crosslink thiol groups, amino-thiol crosslinking agents that crosslink amino groups and thiol groups, carboxyl-amino crosslinking agents that crosslink carboxyl groups and amino groups, hydroxy-thiol crosslinking agents that crosslink hydroxyl groups and thiol groups, etc. can be cited. The primary amino group exists at the N-terminus of the target protein, the antifreeze protein, and the side chain of lysine, and is positively charged at physiological pH. Therefore, it mainly exists on the outer surface of the three-dimensional structure of the target protein. Therefore, from the aspect of crosslinking between such primary amino groups suitable for existing on the outer surface, an amine-reactive crosslinking agent is preferably used as the protein crosslinking agent.

[0022] Examples of the amine-reactive crosslinking agent include crosslinking agents having an N-hydroxy ester (NHS ester) group as a reactive group such as N,N'-disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), bis(3-sulfo-N-succinimidyl)suberate (BS3), and salts thereof; crosslinking agents having an imido ester group as a reactive group such as dimethyl adipate (DMA), dimethyl pimelate (DMP), dimethyl suberate (DMS); crosslinking agents having an aldehyde group as a reactive group such as formaldehyde and glutaraldehyde. Among them, as the amine-reactive crosslinking agent, glutaraldehyde, or bis(3-sulfo-N-succinimidyl)suberate or a salt thereof is preferred, and bis(3-sulfo-N-succinimidyl)suberate or a salt thereof is more preferred from the aspect of not causing a polymerization reaction between crosslinking agents.

[0023] [Expression vector] Instead of or in addition to the above-mentioned antifreeze protein and protein crosslinking agent, the preparation kit of the present embodiment may include an expression vector containing a nucleic acid encoding an antifreeze protein. Examples of the antifreeze protein include the same substances as those exemplified in the above "antifreeze protein". Examples of the nucleic acid encoding an antifreeze protein include a nucleic acid encoding the HPLC6 peptide. The nucleic acid encoding the HPLC6 peptide (SEQ ID NO: 2) is a partial nucleotide sequence of the nucleic acid encoding the antifreeze protein from Pseudopleuronectes americanus. The full-length nucleotide sequence of the antifreeze protein from Pseudopleuronectes americanus is described, for example, in GenBank accession numbers AH005322, X07506, M62414, M62416, M62417, etc.

[0024] There is no particular limitation on the expression vector for inserting the above nucleic acid. For example, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, phages, virus vectors, and vectors obtained by modifying them can be used. Examples of plasmids from Escherichia coli include pBR322, pBR325, pUC12, and pUC13. Examples of plasmids from Bacillus subtilis include pUB110, pTP5, pC194, etc. Examples of plasmids from yeast include pSH19, pSH15, etc. An example of a phage is λ phage. Examples of viruses that are the source of virus vectors include adenovirus, adeno-associated virus, lentivirus, vaccinia virus, baculovirus, retrovirus, hepatitis virus, etc.

[0025] Among the above expression vectors, there is no particular limitation on the promoter for expressing the antifreeze protein. It can be a promoter for expression using animal cells as a host, a promoter for expression using plant cells as a host, or a promoter for expression using insect cells as a host. Examples of promoters for expression using animal cells as a host include EF1α promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-tk promoter, CAG promoter, etc. Examples of promoters for expression using plant cells as a host include the 35S promoter of cauliflower mosaic virus (CaMV), REF (rubber elongation factor) promoter, etc. Examples of promoters for expression using insect cells as a host include polyhedrin promoter, p10 promoter, etc. These promoters can be appropriately selected according to the type of host for expressing the antifreeze protein.

[0026] The above expression vector may also have a multiple cloning site, enhancer, splicing signal, polyadenylation signal, selection marker, origin of replication, etc.

[0027] In the above expression vector, it is preferable to add a separate target gene (in this embodiment, a gene encoding a target protein) upstream or downstream of the nucleic acid encoding the antifreeze protein. Alternatively, in the above expression vector, the nucleic acid encoding the antifreeze protein may also be in a form inserted into the middle of the base sequence of the gene encoding the target protein. At this time, by appropriately adjusting the positional relationship between the nucleic acid encoding the antifreeze protein and the gene encoding the target protein in the expression vector, a fusion protein in which the antifreeze protein is bound to the desired position of the target protein can be prepared. That is, the insertion position of the nucleic acid encoding the antifreeze protein can be appropriately selected according to the secondary structure of the target protein. For example, it can be set to insert the antifreeze protein at the part of the loop connecting the α-helix structure and the β-sheet.

[0028] As described above, the above-described expression vector can produce a fusion protein in the form of a fusion of an antifreeze protein and a target protein by having a nucleic acid encoding an antifreeze protein and a gene encoding a target protein. In addition, in the case of an expression vector incorporating a protein secretion signal to the outside of the cell, a fusion protein of the target protein in the form of a protein secretion signal and an amino acid sequence to which an antifreeze protein is attached can be generated and recovered in the culture solution. In addition, in the case of an intracellular expression vector, the same antifreeze protein-added fusion protein can also be generated.

[0029] As described above, by using an expression vector into which a nucleic acid encoding an antifreeze protein and a gene encoding a target protein are inserted and an appropriate host cell corresponding to the type of the expression vector, a fusion protein of the above-described antifreeze protein and the target protein can be expressed.

[0030] <Second Embodiment> A sample preparation kit for cryo-electron microscopy according to an embodiment of the present invention (hereinafter, sometimes referred to as "the preparation kit of the present embodiment") includes an ice thickness control molecule to which an antifreeze protein is bound.

[0031] In cryo-electron microscopy, in order to record a high-resolution particle image with high image quality while suppressing damage as much as possible, it is necessary to embed the target protein in an amorphous ice film by rapidly freezing an aqueous solution of the target protein into a uniform thin film. However, depending on the type of the target protein, it sometimes concentrates in the central part of the observation area of the grid or aggregates at the edge of the observation area, resulting in non-uniform ice thickness and relatively thick ice formation.

[0032] In contrast, according to the preparation kit of the present embodiment, the ice thickness control molecule having an antifreeze protein bound thereto and having a certain thickness or particle size is uniformly dispersed inside the observation area (hole) of the grid according to the property of the antifreeze protein to aggregate at the gas-liquid interface. Thus, according to the thickness or particle size of the ice thickness control molecule, uniform ice can be formed even with a thickness of about several tens of nm.

[0033] [Ice Thickness Control Molecule] As the ice thickness control molecule, a polymer satisfying the following conditions can be used. 1) The particle size of the ice thickness control molecule is slightly larger than the particle size of the target protein. 2) The shape of the ice thickness control molecule is different from the shape of the target protein and is easily distinguishable. 3) It can bind to an antifreeze protein.

[0034] Specific examples of such an ice thickness control molecule include polyproteins or heteroproteins, but are not limited thereto.

[0035] Among them, as the ice thickness control molecule, a multimeric protein can preferably be used.

[0036] As the multimeric protein, as long as it is a protein with a known crystal structure and the monomers self-organize in solution to form multimers, there is no particular limitation. The thickness or particle size of the multimeric protein only needs to be of the same size as the desired ice thickness.

[0037] Specific examples of the multimeric protein include, for example, glutamine synthetase (two-layer ring structure, about 16 nm in diameter, about 11 nm in thickness), ferritin (spherical, about 14 nm in particle size), etc.

[0038] These multimeric proteins are obtained by expressing the monomers constituting the multimeric protein and self-organizing them in solution. As the antifreeze protein, the above-mentioned protein cross-linking agent can be used to bind to the multimeric protein, or it can also be bound by expressing a fusion protein of the monomer and the antifreeze protein. Among them, from the aspect of easy adjustment of the binding position of the antifreeze protein, it is preferred to bind it by expressing a fusion protein of the monomer and the antifreeze protein.

[0039] The fusion protein of the monomer and the antifreeze protein can be expressed, for example, by transforming an expression vector containing a nucleic acid encoding the monomer and a nucleic acid encoding the antifreeze protein into an appropriate host cell corresponding to the type of the expression vector. The type of the expression vector and the insertion positions of the nucleic acid encoding the monomer and the nucleic acid encoding the antifreeze protein in the expression vector are as described in the preparation kit of the first embodiment above.

[0040] At this time, the antifreeze protein can bind to all the monomers, or it can also bind to a part of the monomers. That is, the fusion protein of the monomer and the antifreeze protein can be self-organized in solution to prepare a multimeric protein with the antifreeze protein bound to all the monomers, or the fusion protein of the monomer and the antifreeze protein and only the monomers can be self-organized in solution to prepare a multimeric protein with the antifreeze protein bound to a part of the monomers.

[0041] Among them, from the aspect of being able to make the desired surface of the multimeric protein more effectively face the gas-liquid interface and disperse the multimeric protein in the sample in a form with consistent thickness or particle size, it is preferred to bind to all the monomers. That is, the preparation kit of this embodiment can also include an expression vector containing a nucleic acid encoding the monomer constituting the multimeric protein and a nucleic acid encoding the antifreeze protein (or a nucleic acid encoding a fusion protein of the monomer constituting the multimeric protein and the antifreeze protein) instead of the multimeric protein bound with the antifreeze protein.

[0042] As monomers constituting a multimeric protein, specifically, for example, monomers constituting glutamine synthetase (amino acid sequences: for example, refer to GenBank accession numbers CAA28806.1, AAA23879.1, AAB03004.1, AAC76867.1, BAE77439.1, AAA98066.1, AAA23882.1, AAA23880.1; base sequences: for example, refer to GenBank accession numbers X05173, M13746, L19201, U00096, AP009048, J01618, M10421, K02176), monomers constituting ferritin (amino acid sequences: for example, refer to GenBank accession numbers CAA37593.1, AAC74975.1, BAA15728.1, AAA79049.1; base sequences: for example, refer to GenBank accession numbers X53513, U00096, AP009048, U35066), etc. can be cited.

[0043] In addition, as a nucleic acid encoding a fusion protein of a monomer constituting a multimeric protein and an antifreeze protein, for example, a nucleic acid encoding a fusion protein in which an HPLC6 peptide, which is a type I antifreeze protein, is bound as an antifreeze protein to the N-terminus of a monomer of glutamine synthetase, a nucleic acid encoding a fusion protein in which an HPLC6 peptide, which is a type I antifreeze protein, is bound as an antifreeze protein to the N-terminus of a monomer of ferritin, etc. can be cited.

[0044] <Other structures> In addition to the above-described constitution, the preparation kit of the present embodiment may further include a host cell corresponding to the type of expression vector, a known buffer (such as phosphate-buffered saline (PBS)) for dissolving or dispersing the target protein, a membrane hole grid usually used in a sample for cryo-electron microscopy, etc.

[0045] In addition, the preparation kit of the present embodiment may be used in combination with the preparation kit for a sample for cryo-electron microscopy according to the first embodiment and the preparation kit for a sample for cryo-electron microscopy according to the second embodiment.

[0046] That is, the preparation kit according to the first embodiment of the present invention may further include a multimeric protein bound to an antifreeze protein.

[0047] Alternatively, the preparation kit according to the second embodiment of the present invention may further include an antifreeze protein and a protein crosslinking agent, or an expression vector containing a nucleic acid encoding an antifreeze protein.

[0048] Accordingly, it is possible to solve three problems: the deviation in the orientation of the target protein in the sample for a cryo-electron microscope with respect to the incident direction of the electron beam, the localization of the adsorption of the target protein to the amorphous carbon film, etc., and the control of the thickness of the ice. That is, by using in combination the preparation kit for a cryo-electron microscope sample according to the first embodiment and the preparation kit for a cryo-electron microscope sample according to the second embodiment, it is possible to obtain a cryo-electron microscope sample in which the deviation in the orientation of the target protein in the sample with respect to the incident direction of the electron beam is suppressed, the target protein is dispersed throughout the observation region, and the thickness of the ice in the sample is uniformly controlled.

[0049] "Preparation Method of Sample for Cryo-Electron Microscope" The preparation method of a sample for a cryo-electron microscope according to one embodiment of the present invention (hereinafter, sometimes referred to as "the preparation method of the present embodiment") includes: using the preparation kit for a cryo-electron microscope sample according to the first embodiment or the second embodiment described above to prepare a cryo-electron microscope sample containing a target protein (hereinafter, sometimes referred to as "the preparation step").

[0050] According to the preparation method of the present embodiment, mainly by using the preparation kit for a cryo-electron microscope sample according to the first embodiment described above, it is possible to suppress the deviation in the orientation of the target protein in the sample with respect to the incident direction of the electron beam. That is, the preparation method of the present embodiment can also be referred to as a method for controlling the orientation of the target protein in a cryo-electron microscope sample. The same applies when the preparation kit of the first embodiment further includes an ice thickness control molecule conjugated with an amphiphilic protein.

[0051] According to the preparation method of the present embodiment, mainly by using the preparation kit for a cryo-electron microscope sample according to the first embodiment described above, it is possible to suppress the collision or adsorption of the target protein with the gas-liquid interface. That is, the preparation method of the present embodiment can also be referred to as a method for suppressing the adsorption of the target protein in a cryo-electron microscope sample to the gas-liquid interface, or a method for suppressing the collision of the target protein in a cryo-electron microscope sample with the gas-liquid interface. The same applies when the preparation kit of the first embodiment further includes an ice thickness control molecule conjugated with an amphiphilic protein.

[0052] According to the preparation method of the present embodiment, mainly by using the preparation kit for a cryo-electron microscope sample according to the first embodiment described above, it is possible to suppress the adsorption of the target protein to the amorphous carbon film in the sample and disperse the target protein throughout the observation region. That is, the preparation method of the present embodiment can also be referred to as a method for suppressing the adsorption of the target protein in a cryo-electron microscope sample to the amorphous carbon film. The same applies when the preparation kit of the first embodiment further includes an ice thickness control molecule conjugated with an amphiphilic protein.

[0053] According to the preparation method of the present embodiment, by mainly using the preparation kit for the specimen for cryo-electron microscopy of the above-described second embodiment, the thickness of the ice of the specimen can be uniformly controlled. That is, the preparation method of the present embodiment can also be referred to as a method for controlling the thickness of the ice of the specimen for cryo-electron microscopy.

[0054] Next, the steps constituting the preparation method of the present embodiment will be described in detail below.

[0055] <Modulation step> In the preparation step, a specimen for cryo-electron microscopy containing the target protein is prepared by using the preparation kit for the specimen for cryo-electron microscopy according to the first embodiment or the second embodiment described above.

[0056] Specifically, for example, in the case of using an antifreeze protein as the preparation kit of the first embodiment, the antifreeze protein and the target protein are mixed in a buffer solution having a pH of 7 or more and 9 or less.

[0057] At this time, the mixing ratio of the target protein and the antifreeze protein may be 1:1 to 1:5 in terms of molar ratio, or may be 1:1 to 1:2. By setting the molar ratio within the above range, it is possible to suppress the collision or adsorption of the target protein with the gas-liquid interface, and as a result, it is possible to further suppress the deviation of the posture of the target protein with respect to the electron beam incident direction, and project the target proteins in various postures.

[0058] In addition, for example, in the case of using a protein crosslinking agent and an antifreeze protein in combination as the preparation kit of the first embodiment, first, the target protein and the antifreeze protein are mixed in a solution in the presence of the protein crosslinking agent to bind the antifreeze protein to the target protein.

[0059] At this time, the mixing ratio of the target protein and the antifreeze protein may be 1:10 to 1:200 in terms of molar ratio, or may be 1:20 to 1:120. By setting the molar ratio within the above range, it is possible to more sufficiently bind the antifreeze protein to the target protein, and as a result, it is possible to further suppress the deviation of the posture of the target protein with respect to the electron beam incident direction, and project the target proteins in various postures.

[0060] In addition, the protein crosslinking agent can be used according to a known protocol depending on its type. For example, the amine-reactive crosslinking agent is used at a concentration of about 10 to 50-fold molar amount relative to the total molar amount of the protein in an amine-free buffer at a pH above 7 and below 9. As a specific concentration of the amine-reactive crosslinking agent, it is used at about 0.25 mM or more and 15.00 mM or less in the solution. The crosslinking reaction is carried out at room temperature (about 25°C) for, for example, 1 minute or more and 60 minutes or less, or on ice for 1 hour or more and 5 hours or less. After the reaction, a solution containing a reaction terminator is added, or the unreacted amine-reactive crosslinking agent is removed by dialysis or desalting to terminate the crosslinking reaction.

[0061] Alternatively, for example, in the case of using an expression vector containing a nucleic acid encoding an antifreeze protein as the preparation kit of the first embodiment above, a gene encoding the target protein is inserted at a desired position in the expression vector. Regarding the insertion positions of the nucleic acid encoding the antifreeze protein and the gene encoding the target protein in the expression vector, it is as described in the preparation kit of the first embodiment above. Next, the expression vector containing the nucleic acid encoding the antifreeze protein and the gene encoding the target protein is transformed into an appropriate host cell corresponding to the type of the expression vector, whereby a fusion protein of the antifreeze protein and the target protein is expressed. The fusion protein is obtained from the culture solution of the host cell or from inside the host cell by disrupting the host cell. If necessary, the obtained fusion protein can be purified by a known method.

[0062] Or, for example, in the case of using the preparation kit of the second embodiment, the multimeric protein conjugated with the antifreeze protein and the target protein are mixed in an amine-free buffer at a pH above 7 and below 9. The target protein can be used directly, or a substance conjugated with the antifreeze protein using the preparation kit of the first embodiment above can also be used.

[0063] The respective concentrations of the multimeric protein conjugated with the antifreeze protein and the target protein contained in the mixed solution of the multimeric protein conjugated with the antifreeze protein and the target protein can be appropriately adjusted according to the type of the multimeric protein used. For example, when the multimeric protein is ferritin, it can be 0.1 μM or more and 3.0 μM or less, or it can be 0.5 μM or more and 1.5 μM or less. In addition, at this time, the concentration of the target protein contained in the mixed solution can be 1.0 μM or more and 5.0 μM or less, or it can be 2.0 μM or more and 4.0 μM or less. By setting the concentrations of these proteins within the above ranges, uniform ice corresponding to the thickness or particle size of the multimeric protein can be formed, and at the same time, the amount of the multimeric protein conjugated with the antifreeze protein present is an amount that does not interfere with the projection of the target protein.

[0064] Next, the solution containing the target protein conjugated with the antifreeze protein or the solution containing the target protein and the multimeric protein conjugated with the antifreeze protein obtained as described above is placed in a membrane pore grid that has been hydrophilized by glow discharge or the like. Next, after removing the excess solution using filter paper or the like, the grid is rapidly dropped into liquid ethane to freeze the sample. Thus, a frozen grid in which the target protein is encapsulated in a film of amorphous thin ice is obtained. The obtained frozen grid is stored in liquid nitrogen until observation using a cryo-electron microscope.

[0065] The sample for cryo-electron microscopy obtained by the preparation method of the present embodiment is mounted on a cryo-transfer holder or a dedicated cassette in a cryo-workstation cooled with liquid nitrogen and inserted into the cryo-electron microscope in a non-frosting manner for imaging. Structural analysis of the target protein based on single particle analysis can be performed based on the imaged data. In addition, it can also be applied to structural analysis using sub-tomogram averaging in electron beam tomography.

[0066] As described above, each embodiment using the antifreeze protein (or a vector containing a nucleic acid encoding the antifreeze protein) has been described in detail. However, the preparation kit of each of the above embodiments may also include an amphiphilic protein other than the antifreeze protein (or a vector containing a nucleic acid encoding the amphiphilic protein) instead of the antifreeze protein (or a vector containing a nucleic acid encoding the antifreeze protein), or together with the antifreeze protein. In addition, in the preparation method of each of the above embodiments, an amphiphilic protein other than the antifreeze protein (or a vector containing a nucleic acid encoding the amphiphilic protein) can be used instead of the antifreeze protein (or a vector containing a nucleic acid encoding the antifreeze protein), or together with the antifreeze protein.

[0067] The amphiphilic protein has a hydrophobic-rich region and a hydrophilic-rich region. Thus, the hydrophobic-rich region of the amphiphilic protein faces the air side, and the hydrophilic-rich region faces the water side. Therefore, the amphiphilic protein is not limited to the antifreeze protein and has the property of aggregating at the air-liquid interface. Therefore, even when an amphiphilic protein other than the antifreeze protein is used, the same effect can be obtained.

[0068] As the amphiphilic protein, an amphiphilic protein having a small and relatively simple structure is preferred, and thus an amphiphilic protein having an amphiphilic α-helical structure is particularly preferred. As such proteins, for example, type I antifreeze proteins such as HPLC6 shown in the examples, KALA peptides, etc. can be cited, but are not limited to these. [Examples]

[0069] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

[0070] [Example 1] (Binding of Antifreeze Protein and Target Protein Using Protein Crosslinker) An attempt was made to control the orientation of the target protein with respect to the electron beam incident direction by binding the antifreeze protein to the amino groups on the surface of the target protein using a protein crosslinker.

[0071] 1. Crosslinking Reaction of HPLC6 and Target Protein As the target protein, hemagglutinin (HA) protein (manufactured by MyBioSource, MBS434205) was used. As the antifreeze protein, the HPLC6 peptide (amino acid sequence: SEQ ID NO: 1) as a type I antifreeze protein was expressed as a GST fusion protein in Escherichia coli and then purified by cleaving GST for use. The HA protein (concentration: 1 mg / mL) dissolved in PBS and the HPLC6 peptide (concentration: 1.7 mg / mL) were mixed in a molar ratio of 1:0, 1:4, 1:22, or 1:110, and crosslinked at room temperature (about 25 °C) for 5 minutes in the presence of 0.14% by mass of glutaraldehyde as a protein crosslinker. 1 M Tris-HCl buffer (pH 8.0) was added to make the final concentration 44 mM, and after stopping the crosslinking reaction, each sample solution was prepared (concentration of HA protein: 1.6 μM (the concentration of HA protein was the same in each sample, and the concentration of HPLC6 peptide was different)).

[0072] 2. Preparation of Specimen Grids After subjecting a membrane hole grid (Quantifoil R1.2 / 1.3 Cu 300) to hydrophilic treatment by glow discharge, 3 μL of the sample solution prepared in the above “1.” was placed on the grid. Then, after removing the excess solution with filter paper, the grid was quickly dropped into liquid ethane to freeze the sample. Thus, a frozen grid in which the protein sample was encapsulated in an amorphous thin ice film was obtained. The obtained frozen grid was stored in liquid nitrogen until observation with a cryo-electron microscope.

[0073] 3. Observation with Cryo-Electron Microscope Each frozen grid prepared in the above “2.” was inserted into a cryo-electron microscope (Talos Arctica manufactured by Thermo Fisher Scientific) and observed and photographed at an accelerating voltage of 200 kV. From the photographed images, particle images projected in various orientations were cut out using RELION, classified into particle images with the same orientation, arranged, and then the orientations and positions of the particle images classified by each projection direction were made consistent to obtain an average image. The results are shown in Figure 1 . In Figure 1Among them, the number recorded horizontally for the molar ratio of HA protein to HPLC6 is the total number of projected particle images. In addition, the number recorded on the particle image represents the number of particle images in the same orientation, and the particle images are arranged from left to right in order starting from the particle image with the largest number of particle images. In addition, in Figure 1 Among them, the symbol "T" represents a particle image of HA protein projected from above, and the symbol "S" represents a particle image of HA protein projected horizontally.

[0074] As Figure 1 shown, in the molar ratio of HA protein to HPLC6 peptide, as the molar ratio of HPLC6 peptide increases, there is a tendency for the number of particle images of HA protein projected horizontally to increase. When the molar ratio of HA protein to HPLC6 peptide is 1:0 to 1:22, the number of particle images of HA protein projected from above is the largest. When the molar ratio of HA protein to HPLC6 peptide is 1:110, the number of particle images of HA protein projected horizontally is the largest. It is speculated that by increasing the molar ratio of HPLC6 peptide, the amount of HPLC6 peptide bound to the lysine residue or the amino group at the N-terminus existing laterally (side) of the HA protein increases, and the surface (side) to which this HPLC6 peptide binds faces the gas-liquid interface, resulting in an increase in the particle images of HA protein projected horizontally.

[0075] Therefore, it was confirmed that in the molar ratio of the target protein to HPLC6 peptide, by increasing the molar ratio of HPLC6 peptide, the posture of the target protein relative to the electron beam incident direction can be controlled, and a large number of particle images projected in each direction can be collected.

[0076] [Example 2] (Antifreeze protein's inhibitory effect on the adsorption of target protein to amorphous carbon film) For a target protein having the property of being easily adsorbed to the amorphous carbon film in the grid, it was studied to inhibit the adsorption to the amorphous carbon film by genetically engineering the preparation of a fusion protein of an antifreeze protein and the target protein, and to disperse the target protein throughout the observation area.

[0077] 1. Expression and purification of fusion protein As the target protein, glutamine synthetase was used. Glutamine synthetase is composed of a homo-12-mer and has a hexagonal prism-like shape with a diameter of about 16 nm and a height (thickness) of about 11 nm. The monomers of glutamine synthetase self-organize in solution to form a 12-mer. A nucleic acid encoding a fusion protein in which an HHPLC6 peptide (amino acid sequence: SEQ ID NO: 1), which is a type I antifreeze protein, is bound to the N-terminus of the monomer of glutamine synthetase (amino acid sequence: SEQ ID NO: 3) via a linker sequence (amino acid sequence: SEQ ID NO: 4; base sequence: SEQ ID NO: 5) was inserted into the plasmid vector pET-28a (manufactured by Novagen). This vector was transformed into Escherichia coli BL21(DE3) strain. The target protein was purified from the soluble fraction of the induced Escherichia coli using a nickel column, and after cleavage of the His tag, it was further purified using a gel filtration column. In addition, as a control, a nucleic acid (base sequence: SEQ ID NO: 6) encoding only the monomer of glutamine synthetase without binding an antifreeze protein was inserted into a plasmid vector, and using an Escherichia coli expression system, expression and purification were carried out in the same manner.

[0078] 2. Preparation of specimen grids Dilution was carried out with a buffer (composition: 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2). For the specimen containing glutamine synthetase bound with the HPLC6 peptide, the protein concentration was made 2.0 μM, and for the specimen containing only glutamine synthetase, the protein concentration was made 2.0 μM. Using the same method as in “2.” of Example 1, specimen grids were prepared.

[0079] 3. Observation using a cryo-electron microscope Each frozen grid prepared in the above “2.” was inserted into a cryo-electron microscope (Talos Arctica, manufactured by Thermo Fisher Scientific), and observation and imaging were carried out at an accelerating voltage of 200 kV. The results are shown in Figure 2A (glutamine synthetase only) and Figure 2B (glutamine synthetase bound with the HPLC6 peptide).

[0080] As Figure 2A shown, in the specimen grid containing only glutamine synthetase, glutamine synthetase was not observed in the center of the pores, and glutamine synthetase was adsorbed to the amorphous carbon film on the pore periphery and was not dispersed throughout the observation area.

[0081] On the other hand, as Figure 2B shown, in the specimen grid containing glutamine synthetase bound with the HPLC6 peptide, the particles were dispersed without omission in both the center and the periphery of the pores. In addition, inFigure 2B Among them, particle images of the 12-mer of glutamine synthetase projected from above were mainly observed. It is speculated that this is because, in the sample, monomers of glutamine synthetase with the HPLC6 peptide bound to the N-terminus form a 12-mer, and the surface (upper surface) to which the HPLC6 peptide is bound faces the gas-liquid interface. As a result, the postures of the 12-mer of glutamine synthetase with respect to the electron beam incident direction are consistent, and particle images projected from above are mainly observed. Furthermore, it was confirmed that in the overall image of the hole, the inside of the hole was whitened overall, and uniform and thin ice was formed.

[0082] Therefore, it was confirmed that by making a target protein having the property of being easily adsorbed to the amorphous carbon film in the grid into a fusion protein with an antifreeze protein, adsorption to the amorphous carbon film can be inhibited, and particles of the target protein can be dispersed throughout the observation region.

[0083] In addition, it was found that by using multimeric proteins with the same thickness and particle size, the thickness of the ice in the grid can be controlled to be thin and uniform.

[0084] [Example 3] (Control of Ice Thickness by Multimeric Proteins Bound with Antifreeze Proteins) It was investigated whether the thickness of the ice in the grid can be controlled to be thin and uniform by making a fusion protein bound with an antifreeze protein in multimeric proteins with the same thickness and particle size other than glutamine synthetase.

[0085] 1. Expression and Purification of Fusion Protein As the target protein, ferritin was used. In addition, in Example 3, ferritin serves both as a target protein and as an ice thickness control molecule. Ferritin is composed of homo-24-mers and is a spherical shape with a diameter of about 14 nm. Monomers of ferritin self-organize in solution to form 24-mers. A nucleic acid encoding a fusion protein, in which the HHPLC6 peptide (amino acid sequence: SEQ ID NO: 1), which is a type I antifreeze protein, is bound via a linker sequence (amino acid sequence: SEQ ID NO: 8; base sequence: SEQ ID NO: 9) to the N-terminus of a monomer of ferritin (amino acid sequence: SEQ ID NO: 7), was inserted into the plasmid vector pET-28a (manufactured by Novagen). This vector was transformed into Escherichia coli. The target protein was purified from the soluble fraction of the Escherichia coli induced for expression using a nickel column, and further purified using a gel filtration column after cleaving the His tag. In addition, as a control, a nucleic acid (base sequence: SEQ ID NO: 10) encoding only the monomer of ferritin without binding an antifreeze protein was inserted into the plasmid vector, and an Escherichia coli expression system was used for expression and purification in the same manner.

[0086] 2. Preparation of Sample Grid Dilute with a buffer solution (composition: 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2). For the sample containing ferritin bound to the HPLC6 peptide, adjust the protein concentration to 2 μM, and for the sample containing only ferritin, adjust the protein concentration to 7 μM. Using the same method as in “2.” of Example 1, a sample grid was prepared.

[0087] 3. Observation using cryo-electron microscopy Insert each of the frozen grids prepared in “2.” above into a cryo-electron microscope (Talos Arctica, manufactured by Thermo Fisher Scientific) and observe and photograph at an accelerating voltage of 200 kV. The results are shown in Figure 3 (Left: Ferritin only; Right: Ferritin bound to the HPLC6 peptide).

[0088] As Figure 3 shown, in the sample grid containing only ferritin, only the central part inside the pores is white, and only in the central part is the ice in a thin state. On the other hand, in the sample grid containing ferritin bound to the HPLC6 peptide, the entire inside of the pores is white, and it is confirmed that a uniform and thin ice has formed. It is speculated that this is because in the sample, the monomers of ferritin with the HPLC6 peptide bound to the N-terminus form 24-mers, and the surface (upper surface) bound to the HPLC6 peptide faces the gas-liquid interface and is dispersed throughout the pores. As a result, the thickness of the ice is formed uniformly and thinly according to the diameter of ferritin.

[0089] Therefore, it was confirmed that by using multimeric proteins with consistent thickness and particle size regardless of the type of multimeric protein, the thickness of the ice in the grid can be controlled to be thin and uniform.

[0090] [Example 4] (Control of ice thickness by multimeric proteins conjugated with antifreeze proteins and dispersion effect of target proteins) Regarding target proteins with the property of being prone to aggregation in the central part of the observation area of the grid, it was investigated that by mixing with multimeric proteins conjugated with antifreeze proteins, the thickness of the ice in the grid can be controlled to be thin and uniform, while suppressing the aggregation of the target proteins towards the central part of the observation area of the grid and dispersing the target proteins throughout the observation area.

[0091] 1. Expression and purification of fusion proteins As the target protein, the HA protein (manufactured by My Bio Source, MBS434205) was used. As the fusion protein, a fusion protein in which the HPLC6 peptide, a type I antifreeze protein, was bound as an antifreeze protein to the N-terminus of the monomer of ferritin was expressed and purified by the same method as in "1." of Example 3 above.

[0092] 2. Preparation of sample grids For the sample containing the HA protein and the ferritin fused with the HPLC6 peptide, it was diluted with a buffer (composition: 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2) so that the concentration of the HA protein was 3.0 μM and the concentration of the ferritin fused with the HPLC6 peptide was 1.0 μM, and sample grids were prepared using the same method as in "2." of Example 1.

[0093] 3. Observation using cryo-electron microscopy Each frozen grid prepared in "2." above was inserted into a cryo-electron microscope (Talos Arctica manufactured by Thermo Fisher Scientific) and observed and photographed at an accelerating voltage of 200 kV. The results are shown in Figure 4 .

[0094] As Figure 4 shown in the left image of, it was confirmed that the inside of the pores was entirely white and uniform and thin ice was formed. In addition, as Figure 4 shown in the right image of, the HA protein was dispersed throughout the observation area.

[0095] Therefore, it was confirmed that by combining the use of the target protein and the multimeric protein bound with the antifreeze protein, it was possible to control the thickness of the ice on the grid to be thin and uniform while dispersing the target protein throughout the observation area.

[0096] [Example 5] (Suppression of deviation of posture by suppressing the adsorption of the target protein to the gas-liquid interface by protection at the gas-liquid interface of the antifreeze protein.) Regarding the target protein having the property of easily adsorbing to the gas-liquid interface in a specific posture, it was investigated to perform gas-liquid interface protection by mixing with the antifreeze protein to suppress the collision of the target protein with the gas-liquid interface and adsorption, thereby suppressing the deviation of the posture of the target protein.

[0097] 1. Preparation of the target protein and the antifreeze protein As the target protein, a mutant Y12A of Inosine-5’-monophosphate dehydrogenase 2 (IMPDH2) (hereinafter sometimes referred to as “IMPDH2(Y12A)”) was used. IMPDH2(Y12A) was prepared by expressing it in Escherichia coli after introducing the Y12A mutation into a plasmid containing the gene encoding wild-type IMPDH2. As the antifreeze protein, HPLC6 peptide (amino acid sequence: SEQ ID NO: 1), which is a type I antifreeze protein, was expressed as a GST fusion protein in Escherichia coli and then purified by cleaving GST for use.

[0098] 2. Preparation of Sample Grids For the sample containing IMPDH2(Y12A) and HPLC6 peptide, it was diluted with a buffer (composition: 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2) so that the molar ratio of IMPDH2(Y12A) to HPLC6 peptide became 1:1.25, and sample grids were prepared using the same method as “2.” in Example 1. As the grids, a membrane pore grid (Quantifoil R1.2 / 1.3Cu 300) and a graphene grid (manufactured by Air Membrane Co., Ltd., 2-layer graphene TEM grid Quantifoil R1.2 / 1.3 300mesh Au after hydrophilic treatment) were used. As a control, a sample containing only the target protein was also prepared.

[0099] 3. Observation Using Cryo-Electron Microscopy Each frozen grid prepared in “2.” above was inserted into a cryo-electron microscope (Talos Arctica, manufactured by Thermo Fisher Scientific) and observed and photographed at an accelerating voltage of 200 kV. The results are shown in Figure 5 . In Figure 5 , the symbol “T” represents the particle image of IMPDH2(Y12A) projected from above, and the symbol “S” represents the particle image of IMPDH2(Y12A) projected from the side.

[0100] As shown in (A) and (C) of Figure 5 , in the sample containing only IMPDH2(Y12A) as the target protein, it adsorbed to the gas-liquid interface in a specific posture, and the number of particle images of IMPDH2(Y12A) projected from above was in the majority. On the other hand, as shown in (B) and (D) of Figure 5 , in the sample containing IMPDH2(Y12A) and HPLC6 peptide, particle images of IMPDH2(Y12A) projected from the side or obliquely from above were also observed.

[0101] Therefore, it was confirmed that by using the target protein in combination with the HPLC6 peptide, it was possible to inhibit the adsorption of the target protein by collision with the gas-liquid interface, and it was possible to collect a large number of particle images projected in all directions.

[0102] [Example 6] (Inhibitory effect of amphiphilic proteins other than antifreeze proteins on the adsorption of target proteins to amorphous carbon films) For target proteins with properties that are easily adsorbed to the amorphous carbon film in the grid, it was investigated that by preparing a fusion protein of an amphiphilic protein other than the antifreeze protein and the target protein, the adsorption to the amorphous carbon film was inhibited, and the target protein was dispersed throughout the observation area.

[0103] 1. Expression and purification of the fusion protein As the target protein, ferritin was used. The nucleic acid encoding the fusion protein was inserted into the plasmid vector pET-28a (manufactured by Novagen), and the fusion protein was formed by binding a protein consisting of the amino acid sequence (SEQ ID NO: 11) containing the KALA peptide, which is an example of an amphiphilic protein other than the antifreeze protein, to the N-terminus of the monomer of ferritin. In this fusion protein, the monomer of ferritin and the protein containing the KALA peptide are linked by a linker sequence (amino acid sequence: SEQ ID NO: 8). This vector was transformed into Escherichia coli BL21(DE3) strain. The target protein was purified from the soluble fraction of the induced Escherichia coli using a nickel column, and after cleaving the His tag, it was further purified using a gel filtration column. In addition, as a control, a sample of only ferritin obtained from the Escherichia coli expression system using a sequence without an amphiphilic protein, which was detailed in "1." of Example 3, was also prepared.

[0104] 2. Preparation of the sample grid For the sample containing ferritin bound to the KALA peptide and the sample containing only ferritin, they were diluted with a buffer (composition: 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2) so that the protein concentration was 2.0 μM, and the frozen grid of the sample was prepared using the same method as in "2." of Example 1.

[0105] 3. Observation using a cryo-electron microscope Each frozen grid prepared in the above "2." was inserted into a cryo-electron microscope (Talos Arctica, manufactured by Thermo Fisher Scientific), and observed and photographed at an accelerating voltage of 200 kV.

[0106] Figure 6A is an image of ferritin taken with a cryo-electron microscope in Example 6,Figure 6B This is an image of the fusion protein of ferritin and KALA peptide taken by cryo-electron microscopy in Example 6.

[0107] As Figure 6A shown, in the frozen grid containing only ferritin, ferritin was not observed in the center of the pores, and ferritin was adsorbed on the amorphous carbon film around the pores, and ferritin was not dispersed throughout the observation area.

[0108] In contrast, as Figure 6B shown, in the frozen grid containing ferritin bound to KALA peptide, the particles of the fusion protein were dispersed without omission in the center and around the pores indicated by the arrows.

[0109] From the above results, it was confirmed that even amphiphilic proteins other than antifreeze proteins can effectively inhibit the adsorption of target proteins to the amorphous carbon film around the pores. It is considered that the present embodiment can be applied to proteins other than HPLC6, such as other amphiphilic α-helical structure-containing proteins, other antifreeze proteins, and other amphiphilic proteins. [Industrial Applicability]

[0110] For the specimen for cryo-electron microscopy according to the present embodiment, deviation of the undesired posture of the target protein in the specimen with respect to the electron beam incident direction, and collision and adsorption to the gas-liquid interface can be suppressed, and a specimen for cryo-electron microscopy in which the target protein is dispersed throughout the observation area can be obtained. In addition, for the specimen for cryo-electron microscopy according to the present embodiment, a specimen for cryo-electron microscopy in which the thickness of the ice in the specimen is uniformly controlled can be obtained. [Explanation of Reference Numerals]

[0111] S, Particle image of the protein projected laterally; T, Particle image of the protein projected from above.

Claims

1. A preparation kit for a specimen for cryo-electron microscopy, which comprises an amphiphilic protein or an expression vector containing a nucleic acid encoding the amphiphilic protein.

2. A preparation kit for a specimen for cryo-electron microscopy, which comprises an ice thickness control molecule conjugated with an amphiphilic protein.

3. The kit for preparing a specimen for a cryo-electron microscope according to claim 1 or 2, wherein, The amphiphilic protein has an amphiphilic α-helical structure.

4. The kit for preparing a specimen for a cryo-electron microscope according to claim 1 or 2, wherein, The amphiphilic protein is an antifreeze protein.

5. The preparation kit for the specimen for cryo-electron microscopy according to claim 4, wherein, The antifreeze protein is a type I antifreeze protein.

6. The kit for preparing a specimen for a cryo-electron microscope according to claim 5, wherein, The type I antifreeze protein is HPLC6 peptide.

7. The kit for preparing a specimen for a cryogenic electron microscope according to claim 1 or 2, wherein, The amphiphilic protein is KALA peptide.

8. The kit for preparing a specimen for a cryo-electron microscope according to claim 1, wherein, It further comprises a protein crosslinking agent.

9. The kit for preparing a specimen for a cryo-electron microscope according to claim 8, wherein, The protein crosslinking agent is an amine-reactive crosslinking agent.

10. The kit for preparing a specimen for a cryogenic electron microscope according to claim 9, wherein, The amine-reactive crosslinking agent is glutaraldehyde or bis(3-sulfo-N-succinimidyl) suberate or a salt thereof.

11. The kit for preparing a specimen for a cryo-electron microscope according to claim 1, wherein, It further comprises an ice thickness control molecule conjugated with an amphiphilic protein.

12. The kit for preparing a specimen for a cryo-electron microscope according to claim 2 or 11, wherein, The ice thickness control molecule is an antifreeze protein and is a multimeric protein.

13. The kit for preparing a specimen for a cryo-electron microscope according to claim 12, wherein, The multimeric protein is glutamine synthetase or ferritin.

14. A method for preparing a specimen for a cryogenic electron microscope, comprising: Use the preparation kit for a specimen for cryo-electron microscopy according to claim 1 or 2 to prepare a specimen for cryo-electron microscopy containing a target protein.

15. A method for controlling the posture of a target protein in a specimen for a cryo-electron microscope, comprising: Use the preparation kit for a specimen for cryo-electron microscopy according to claim 1 or 8 or 11 to prepare a specimen for cryo-electron microscopy containing a target protein.

16. A method for inhibiting the adsorption of a target protein in a specimen for a cryo-electron microscope to an amorphous carbon film, comprising: Use the preparation kit for a specimen for cryo-electron microscopy according to claim 1 or 8 or 11 to prepare a specimen for cryo-electron microscopy containing a target protein.

17. A method for inhibiting the collision and adsorption of a target protein in a sample for a cryo-electron microscope to a gas-liquid interface, comprising: Use the preparation kit for a specimen for cryo-electron microscopy according to claim 1 or 8 or 11 to prepare a specimen for cryo-electron microscopy containing a target protein.

18. A method for controlling the thickness of ice of a specimen for a cryogenic electron microscope, comprising: Use the preparation kit for a specimen for cryo-electron microscopy according to claim 2 or 11 to prepare a specimen for cryo-electron microscopy containing a target protein.

Images