CRYOELECTRON MICROSCOPE SAMPLE PREPARATION KIT AND USE THEREOF

Abstract

The cryo-electron microscope sample preparation kit contains an amphipathic protein and a protein crosslinking agent, or an expression vector containing a nucleic acid sequence encoding the amphipathic protein. The kit also includes an ice thickness control molecule to which an amphipathic protein is attached. The amphipathic protein may have an amphipathic α-helix structure and may be either an antifreeze protein or a type I antifreeze protein.

Description

TECHNICAL AREA

[0001] The present invention relates to a cryo-electron microscope sample preparation kit and its use. In particular, the present invention relates to a cryo-electron microscope sample preparation kit, a preparation method for a cryo-electron microscope sample, a method for controlling the position of a target protein in a cryo-electron microscope sample, a method for suppressing the collision and adsorption of a target protein in a cryo-electron microscope sample to an amorphous carbon film, and a method for controlling the ice thickness in a cryo-electron microscope sample.

[0002] Priority is claimed from Japanese patent application No. 2022-204279, which was filed on December 21, 2022, and the contents of which are hereby incorporated by reference. BACKGROUND OF THE TECHNOLOGY

[0003] In the prior art, X-ray crystallography, NMR spectroscopy, and similar techniques were used to analyze the structure of biological molecules, such as proteins. However, in recent years, single-particle analysis using a cryo-electron microscope (hereinafter referred to simply as the "cryo-electron microscopy technique") has attracted considerable attention. The cryo-electron microscopy technique eliminates the need for sample crystallization, which is essential for X-ray crystallography analysis, and there is no upper limit on the sample's molecular weight that restricts the application of NMR spectroscopy.

[0004] However, since most of the interatomic bonds that maintain the three-dimensional structure of the biological molecule are non-covalent bonds, such as hydrogen bonds or ionic bonds, the biological molecule is more likely to be damaged by electron beam irradiation compared to a sample such as a metal or a semiconductor. Therefore, in order to record a high-resolution projection image (hereafter referred to as a "particle image") of a biological molecular particle with high image quality, the cryo-electron microscopy technique requires... to embed a biological molecule in a thin amorphous ice film by rapidly freezing an aqueous solution of the biological molecule as a thin film, to irradiate the biological molecule with an electron beam while the biological molecule is kept at a low temperature on a sample stage cooled with liquid helium or liquid nitrogen, and to record an electron microscope image.

[0005] Additionally, depending on the type of biological molecule, it may be adsorbed onto the amorphous carbon film of the sample grid and not disperse throughout the entire hole in which the thin amorphous ice film forming the observation area is formed.

[0006] Since each particle image is a projection of a molecule embedded in the thin ice film in various orientations, it is also necessary to collect as many images as possible, projected in different directions and as uniformly as possible, in order to reconstruct the three-dimensional image. This means that it is essential to reduce the noise level and improve the signal level by efficiently collecting as many particle images as possible, aligning the orientation and position of the particle images, and classifying and averaging the particle images for each projection direction. Then, the relative relationship in the projection direction of the particle images is determined and back-projected, making it possible for the first time to reconstruct the three-dimensional image with high resolution.In some cases, however, only a projection image of the particles that have been partially damaged due to collision with the gas-liquid interface, or a projection image obtained by imaging the particles from a specific direction due to adsorption onto the gas-liquid interface, can be obtained.

[0007] On the other hand, the antifreeze protein, which is an amphipathic protein, is a relatively small protein consisting of a polypeptide with approximately 30 or more amino acid residues and 150 or fewer amino acid residues. It is known that the antifreeze protein has the ability to inhibit the freezing of water or a water-containing substance by binding to a specific crystal plane of an ice crystal nucleus and suppressing the growth of the crystal nucleus at temperatures of 0 °C or below. In recent years, it has been shown that the antifreeze protein has the property of accumulating at a gas-liquid interface (see, for example, Non-Patent Document 1 or the like). Citation list Non-patent document

[0008] 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 Technical Problem

[0009] The present invention was made in light of the above circumstances and provides a cryo-electron microscope sample preparation kit that makes it possible to obtain a cryo-electron microscope sample in which undesired positional deviation of the target protein in a sample with respect to an electron beam incidence direction and collision with and adsorption at a gas-liquid interface are suppressed, and in which the target protein is distributed over the entire observation area. Furthermore, a cryo-electron microscope sample preparation kit is provided that makes it possible to obtain a cryo-electron microscope sample in which the ice thickness of a sample is uniformly controlled.Furthermore, a preparation procedure for a cryo-electron microscope sample, a procedure for controlling the position of a target protein in a cryo-electron microscope sample, a procedure for suppressing the adsorption of a target protein in a cryo-electron microscope sample onto an amorphous carbon film, a procedure for suppressing the collision and adsorption of a target protein in a cryo-electron microscope sample onto a gas-liquid interface, and a procedure for controlling the ice thickness in a cryo-electron microscope sample are provided, which utilize the cryo-electron microscope sample preparation kit. Solution to the problem

[0010] That is to say, the present invention comprises the following aspects. (1) Cryo-electron microscope sample preparation kit containing an amphipathic protein or an expression vector containing a nucleic acid sequence encoding the amphipathic protein. (2) Cryo-electron microscope sample preparation kit containing an ice thickness control molecule to which an amphipathic protein is bound. (3) Cryo-electron microscope sample preparation kit according to point (1) or (2) wherein the amphipathic protein has an amphipathic α-helix structure. (4) Cryo-electron microscope sample preparation kit according to one of points (1) to (3) wherein the amphipathic protein is an antifreeze protein. (5) Cryo-electron microscope sample preparation kit according to point (4) wherein the antifreeze protein is a type I antifreeze protein. (6) Cryo-electron microscope sample preparation kit according to point (5) wherein the type I antifreeze protein is an HPLC6 peptide. (7) Cryo-electron microscope sample preparation kit according to one of points (1) to (3) wherein the amphipathic protein is a KALA peptide. (8) Cryo-electron microscope sample preparation kit according to point (1), further comprising a protein crosslinking agent. (9) Cryo-electron microscope sample preparation kit according to point (8) wherein the protein crosslinking agent is an amine-reactive crosslinking agent. (10) Cryo-electron microscope sample preparation kit according to point (9) wherein the amine-reactive crosslinking agent is glutaraldehyde, or bis(3-sulfo-N-succinimidyl)suberate or a salt thereof. (11) Cryo-electron microscope sample preparation kit according to point (1), further comprising an ice thickness control molecule to which an amphipathic protein is bound. (12) Cryo-electron microscope sample preparation kit according to point (2) or (11), wherein the ice thickness control molecule is an antifreeze protein and is a homomultimeric protein. (13) Cryo-electron microscope sample preparation kit according to point (12) wherein the homomultimeric protein is a glutamine synthetase or ferritin. (14) Method of preparation for a cryo-electron microscope sample, comprising preparing a cryo-electron microscope sample containing a target protein using the cryo-electron microscope sample preparation kit according to any of points (1) to (13). (15) Method for controlling the position of a target protein in a cryo-electron microscope sample, the method comprising preparing a cryo-electron microscope sample containing a target protein using the cryo-electron microscope sample preparation kit according to any of points (1), (8) to (11). (16) Method for suppressing the adsorption of a target protein in a cryo-electron microscope sample onto an amorphous carbon film, the method comprising preparing a cryo-electron microscope sample containing a target protein using the cryo-electron microscope sample preparation kit according to any one of points (1), (8) to (11). (17) Method for suppressing the collision and adsorption of a target protein in a cryo-electron microscope sample at a gas-liquid interface, wherein the method comprises preparing a cryo-electron microscope sample containing a target protein using the cryo-electron microscope sample preparation kit according to any one of points (1), (8) to (11). (18) Method for controlling the ice thickness in a cryo-electron microscope sample, the method comprising preparing a cryo-electron microscope sample containing a target protein using the cryo-electron microscope sample preparation kit according to any one of points (2), (11) to (13). Advantageous effects of the invention

[0011] According to the cryo-electron microscope sample described above, it is possible to obtain a sample in which distortion of an undesired position of the target protein in a sample with respect to the electron beam incidence direction and collision with and adsorption at a gas-liquid interface are suppressed, and in which the target protein is distributed over the entire observation area. Furthermore, according to the cryo-electron microscope sample described above, it is possible to obtain a sample in which the ice thickness of a sample is uniformly controlled. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] A two-dimensional class mean image of a particle image of a hemagglutinin protein to which an HPLC6 peptide is bound, recorded with a cryo-electron microscope in Example 1.

[0013] An image of a glutamine synthetase, taken with a cryo-electron microscope in Example 2.

[0014] An image of a glutamine synthetase to which an HPLC6 peptide is bound, taken with a cryo-electron microscope in Example 2.

[0015] An image of ferritin to which an HPLC6 peptide is bound, taken with a cryo-electron microscope in Example 3.

[0016] An image of a mixed solution of a hemagglutinin protein and ferritin to which an HPLC6 peptide is bound, taken with a cryo-electron microscope in Example 4.

[0017] A two-dimensional class mean image of a particle image of inosine 5'-monophosphate dehydrogenase 2 (IMPDH2), taken with a cryo-electron microscope in Example 5.

[0018] An image of ferritin taken with a cryo-electron microscope in Example 6.

[0019] An image of a fusion protein of ferritin and a KALA peptide, taken with a cryo-electron microscope in Example 6. Description of the embodiments: Cryo-electron microscope sample preparation kit; First embodiment

[0020] The cryo-electron microscope sample preparation kit according to an embodiment of the present invention (hereinafter also referred to as the “preparation kit of the present embodiment”) contains an antifreeze protein or an expression vector containing a nucleic acid sequence encoding the antifreeze protein.

[0021] In cryo-electron microscopy, reconstructing a three-dimensional image of a target protein requires collecting as many images of the protein as possible, projected as uniformly as possible in different directions. However, in the sample grid for a cryo-electron microscope, distortion of the protein's position relative to the electron beam incidence direction can occur, and particle images with the same orientation may constitute the majority. Additionally, the protein may be adsorbed onto an amorphous carbon film with holes (approximately 1 µm in diameter) serving as the grid's observation area and may not be evenly distributed within the observation area, resulting in an insufficient number of particle images.Furthermore, in some cases only a projection image of the particles, which were partially damaged due to the collision with the gas-liquid interface, or a projection image obtained by imaging the particles from a specific direction due to adsorption onto the gas-liquid interface, can be obtained.

[0022] On the other hand, in the preparation kit of the present embodiment, due to the property of the antifreeze protein accumulating at the gas-liquid interface, the target protein, which has the property of being readily adsorbed onto the gas-liquid interface in a specific position, is mixed with the antifreeze protein to protect the gas-liquid interface. This prevents the target protein from colliding with or adsorbing onto the gas-liquid interface. As a result, it is possible to reduce particle images of partially damaged target proteins and to decrease and eliminate the preferential orientation of target protein particles. In other words, it is possible to suppress the distortion of the protein's position relative to the direction of incidence of the electron beam and to obtain projection images of proteins in various positions.

[0023] Since the antifreeze protein also tends to accumulate at the air-liquid interface, the preparation kit of this embodiment can suppress the adsorption of the target protein onto the amorphous carbon film and distribute the target protein evenly within the observation area (hole) of the grid.

[0024] It is noted that in the present description, the cryo-electron microscopy method means a process in which a sample solution of a biological molecule, such as an isolated and purified protein, is rapidly frozen to embed the biological molecule in a thin film of ice, several hundred to several thousand images are obtained using a special transmission electron microscope (TEM), which can perform an observation by irradiating the sample with an electron beam while the sample is kept at a low temperature under cooling with liquid nitrogen (approximately -196 °C), and a three-dimensional structure of a biological target molecule is reconstructed from the obtained electron microscope image by computer image processing.

[0025] In this description, the cryo-electron microscope sample is a sample prepared by rapidly freezing a solution containing a target protein onto a film-perforated grid. A film-perforated grid used in a cryo-electron microscope has a structure in which a support film with regularly spaced holes is mounted on a metal mesh with holes arranged in a grid pattern and made of a conductive material such as copper, gold, or molybdenum. A thin carbon film of approximately 10 nm or more and approximately 50 nm or less is typically used as the support film, but the thin carbon film coated with a thin gold film of approximately 5 nm or more and approximately 10 nm or less is also used to prevent the film from becoming charged by electron beam irradiation. Antifreeze protein

[0026] Antifreeze proteins are proteins found in fish and other organisms that live in low-temperature environments. They help sustain life by lowering the freezing point (the temperature at which something freezes) and preventing the recrystallization of ice crystals. Antifreeze proteins bind tightly to the surface of the countless tiny ice crystals that form in water when it freezes, thus inhibiting their growth.

[0027] Antifreeze proteins derived from fish are classified into four types based on their structures. Type I antifreeze is an α-helix protein containing a large number of alanine residues and threonine and aspartic acid residues arranged at regular intervals. Type II antifreeze is a lectin-like C-type protein containing a disulfide bond. Type III antifreeze is a globular protein composed of a characteristic structural motif. Type IV antifreeze contains a large number of glutamine residues and has an unknown three-dimensional structure. Of these, type I antifreeze is preferable because its crystal structure is known, its molecular weight is low, its structure is simple, and it does not interfere with the projection of the target protein's particle image.Examples of the preferred type I antifreeze protein include an HPLC6 peptide consisting of 37 amino acid residues (amino acid sequence: SEQ ID NO: 1), and the like. The HPLC6 peptide represented by SEQ ID NO: 1 has a partial amino acid sequence of an antifreeze protein derived from Pseudopleuronectes americanus. The full-length amino acid sequence of the antifreeze protein derived from Pseudopleuronectes americanus is described, for example, under GenBank accession numbers AB59964.1, CAA30389.1, AAA49469.1, AAA49471.1, AAA49472.1, and the like.

[0028] Additionally, in the preparation kit of the present embodiment, in a case where an expression vector containing a nucleic acid sequence encoding an antifreeze protein is used, the antifreeze protein can be bound to any desired position of the target protein by inserting the nucleic acid sequence encoding the target protein upstream or downstream of the nucleic acid sequence encoding the antifreeze protein. Since antifreeze proteins tend to accumulate at the gas-liquid interface, the surface of the target protein to which the antifreeze protein is bound faces the gas-liquid interface, thus suppressing distortion of the protein's position relative to the electron beam incidence direction, and the projection image of the protein in different positions can be obtained. Protein crosslinking agent

[0029] Alternatively, in the preparation kit of the present embodiment, the antifreeze protein can be bound to any position of the target protein by using the antifreeze protein and the protein crosslinking agent in combination.

[0030] A protein crosslinking agent can be any crosslinking agent generally used to crosslink proteins. Specific examples of protein crosslinking agents include an amine-reactive crosslinking agent that crosslinks between primary amino groups (-NH2), a sulfhydryl-reactive crosslinking agent that crosslinks between sulfhydryl groups, an amino-sulfhydryl crosslinking agent that crosslinks between an amino group and a sulfhydryl group, a carboxyl-amino crosslinking agent that crosslinks between a carboxyl group and an amino group, and a hydroxy-sulfhydryl crosslinking agent that crosslinks between a hydroxy group and a sulfhydryl group.

[0031] The primary amino group is present at the N-terminus or the lysine side chain of the target protein or antifreeze protein and is positively charged at physiological pH. Therefore, the primary amino group is mainly located on the outer surface of the target protein's three-dimensional structure. Consequently, the amine-reactive crosslinking agent is preferred as the protein crosslinking agent because it is suitable for crosslinking the primary amino groups present on the outer surface.

[0032] Examples of amine-reactive crosslinking agents include crosslinking agents with an N-hydroxy ester (NHS) group as the reaction group, such as N,N'-disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), bis(3-sulfo-N-succinimidyl)suberate (BS3), and salts thereof; crosslinking agents with an imide ester group as the reaction group, such as dimethyl adipimidate (DMA), dimethyl pimelimimidate (DMP), and dimethyl suberimidate (DMS); crosslinking agents with an aldehyde group as the reaction group, such as formaldehyde and glutaraldehyde; and the like.

[0033] Among these, glutaraldehyde, bis(3-sulfo-N-succinimidyl)suberate or a salt thereof are preferred as amine-reactive crosslinkers, and bis(3-sulfo-N-succinimidyl)suberate or a salt thereof is even more preferred, since no polymerization reaction takes place between the crosslinkers. Expression vector

[0034] The preparation kit of the present embodiment may contain an expression vector containing a nucleic acid sequence encoding an antifreeze protein, either instead of or in addition to the antifreeze protein and protein crosslinking agent. Examples of the antifreeze protein include the same proteins exemplified in the "antifreeze protein" described above. Examples of the nucleic acid sequence encoding the antifreeze protein include a nucleic acid sequence encoding an HPLC6 peptide, and the like. The nucleic acid sequence (SEQ ID NO: 2) encoding the HPLC6 peptide is a partial base sequence of a nucleic acid sequence encoding an antifreeze protein derived from Pseudopleuronectes americanus.The full-length base sequence of the antifreeze protein originating from Pseudopleuronectes americanus is described, for example, under GenBank accession numbers AH005322, X07506, M62414, M62416, M62417 and the like.

[0035] The expression vector into which the nucleic acid sequence described above is inserted is not particularly restricted, and, for example, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage, a viral vector, a vector obtained by modifying these vectors, and the like can be used. Examples of the plasmid derived from Escherichia coli include pBR322, pBR325, pUC12, pUC13, and the like. Examples of the plasmid derived from Bacillus subtilis include pUB110, pTP5, pC194, and the like. Examples of the plasmid derived from yeast include pSH19, pSH15, and the like. Examples of the bacteriophage include a λ-phage and the like. Examples of the virus from which the viral vector originates include adenovirus, adeno-associated virus, lentivirus, vaccinia virus, baculovirus, retrovirus, hepatitis virus, and the like.

[0036] In the expression vector, the promoter for expressing the antifreeze protein is not particularly restricted and can be a promoter for expression using animal cells as hosts, a promoter for expression using plant cells as hosts, or a promoter for expression using insect cells as hosts. Examples of promoters for expression using animal cells as hosts include an EF1α promoter, an SRα promoter, an SV40 promoter, an LTR promoter, a cytomegalovirus (CMV) promoter, an HSV-tk promoter, a CAG promoter, and the like. Examples of promoters for expression using plant cells as hosts include a 35S promoter of the cauliflower mosaic virus (CaMV), a rubber elongation factor (REF) promoter, and the like.Examples of promoters for expression using insect cells as hosts include a polyhedrine promoter, a p10 promoter, and similar promoters. These promoters can be selected according to the type of host in which the antifreeze protein is expressed.

[0037] The expression vector may also include a multicloning site, an enhancer, a splice signal, a poly(A) addition signal, a selection marker, a replication origin, or the like.

[0038] In the expression vector, a specific target gene (in the present embodiment, a gene encoding a target protein) is preferably added upstream or downstream of the nucleic acid sequence encoding the antifreeze protein. Alternatively, the nucleic acid sequence encoding the antifreeze protein can be inserted into the middle of the base sequence of the gene encoding the target protein in the expression vector. In this case, by appropriately adjusting the positional relationship between the nucleic acid sequence encoding the antifreeze protein in the expression vector and the gene encoding the target protein, it is possible to produce a fusion protein in which the antifreeze protein is bound to a desired position of the target protein.This means that the insertion position of the nucleic acid sequence encoding the antifreeze protein can be appropriately selected according to the secondary structure of the target protein, and, for example, the insertion position can be set so that the antifreeze protein is inserted into a loop segment connecting the α-helix structure and the β-sheet.

[0039] As described above, the expression vector contains a nucleic acid sequence encoding an antifreeze protein and a gene encoding a target protein, allowing the creation of a fusion protein in which the antifreeze protein is fused to the target protein. Additionally, when incorporated into an expression vector with an extracellular protein secretion signal, a fusion protein of the secretion signal and the target protein, to which an amino acid sequence of the antifreeze protein has been added, can be generated and isolated in a culture solution. Furthermore, the same antifreeze protein-added fusion protein can also be generated using an intracellular expression vector.

[0040] As described above, a fusion protein of the antifreeze protein described above and the target protein can be expressed using an expression vector into which a nucleic acid sequence encoding an antifreeze protein and a gene encoding a target protein are inserted, and a suitable host cell according to the type of expression vector. Second embodiment

[0041] The cryo-electron microscope sample preparation kit according to an embodiment of the present invention (hereinafter also referred to as the "preparation kit of the present embodiment") contains an ice thickness control molecule to which the antifreeze protein is bound.

[0042] In cryo-electron microscopy, to minimize damage and record a high-resolution particle image with high image quality, it is necessary to rapidly freeze an aqueous solution of the target protein as a uniform, thin film, embedding the target protein in a thin amorphous ice film. However, depending on the type of target molecule, aggregation can occur in the center or at the edge of the observation area of ​​the grating, leading to uneven ice thickness and the formation of a thick ice film.

[0043] On the other hand, according to the preparation kit of the present embodiment, the ice thickness control molecules, to which the antifreeze protein is bound and which have a certain thickness or particle diameter, are uniformly distributed within the observation area (hole) of the grid due to the property that the antifreeze protein accumulates at the gas-liquid interface, making it possible to form uniform ice with a thickness of several tens of nm, depending on the thickness or particle diameter of the ice thickness control molecule. Ice thickness control molecule

[0044] A polymer that meets the following requirements can be used as an ice thickness control molecule. 1) The particle diameter of the ice thickness control molecule is slightly larger than the particle diameter of the target protein. 2) The shape of the ice thickness control molecule differs from the shape of the target protein, making it easy to distinguish. 3) The protein can bind to an antifreeze protein.

[0045] Specific examples of such an ice thickness control molecule include a homo- or heteromultimeric protein and the like, but the present invention is not limited thereto.

[0046] Among these, a homomultimeric protein can preferably be used as the ice thickness control molecule.

[0047] The homomultimeric protein is not particularly restricted, as long as it is a protein with a known crystal structure and the monomers self-assemble in solution to form a multimer. The thickness or particle diameter of the homomultimeric protein simply needs to be the same as the desired ice thickness.

[0048] Specific examples of homomultimeric proteins include glutamine synthetase (a double-ring structure, a diameter of about 16 nm, a thickness of about 11 nm), ferritin (spherical, a particle diameter of about 14 nm), and the like.

[0049] These homomultimeric proteins are obtained by expressing a monomer that forms the homomultimeric protein and allowing the monomer to self-assemble in solution. The antifreeze protein can be attached to the homomultimeric protein using the protein crosslinking agent described above, or it can be attached by expressing a fusion protein consisting of a monomer and the antifreeze protein. Of these two methods, attaching the monomer and the antifreeze protein by expressing a fusion protein is preferable because the binding position of the antifreeze protein is easily adjustable.

[0050] The fusion protein consisting of a monomer and an antifreeze protein can be expressed, for example, by transforming a suitable host cell with an expression vector containing a nucleic acid sequence encoding the monomer and a nucleic acid sequence encoding the antifreeze protein, according to the type of expression vector. The type of expression vector and the insertion positions of the nucleic acid sequence encoding a monomer and the nucleic acid sequence encoding an antifreeze protein in the expression vector are as described in the preparation kit of the first embodiment.

[0051] In this case, the antifreeze protein can be bound to all monomers or to a subset of them. That is, a homomultimeric protein in which the antifreeze protein is bound to all monomers can be prepared by self-assembly of the fusion protein from the monomer and the antifreeze protein in a solution, or a homomultimeric protein in which the antifreeze protein is bound to a subset of the monomers can be prepared by self-assembly of the fusion protein from the monomer and the antifreeze protein, or from only the monomer, in a solution.

[0052] Among these, it is preferable for the antifreeze protein to be bound to all monomers, since the homomultimeric protein can be distributed in the sample in a manner where the thickness or particle diameter is more uniform by more efficiently directing the desired surface area of ​​the homomultimeric protein towards the air-liquid interface. That is to say, the preparation kit of the present embodiment can also include an expression vector containing a nucleic acid sequence encoding a monomer forming the homomultimeric protein and a nucleic acid sequence encoding the antifreeze protein (or a nucleic acid sequence encoding a fusion protein of the monomer forming the homomultimeric protein and the antifreeze protein), instead of the homomultimeric protein to which the antifreeze protein is bound.

[0053] Specific examples of the monomer that forms the homomultimeric protein include a monomer that forms glutamine synthetase (see amino acid sequence: for example, GenBank access numbers CAA28806.1, AAA23879.1, AAB03004.1, AAC76867.1, BAE77439.1, AAA98066.1, AAA23882.1 and AAA23880.1 and see base sequence: for example, GenBank access numbers X05173, M13746, L19201, U00096, AP009048, J01618, M10421 and K02176), a monomer that forms ferritin (see amino acid sequence: for example, GenBank access numbers CAA37593.1, AAC74975.1, BAA15728.1 and AAA79049.1 and see base sequence: for example GenBank access numbers X53513, U00096, AP009048 and U35066) and the like.

[0054] Additionally, examples of the nucleic acid sequence encoding the fusion protein consisting of a monomer forming a homomultimeric protein and an antifreeze protein include a nucleic acid sequence encoding a fusion protein as an antifreeze protein in which an HPLC6 peptide, which is a type I antifreeze protein, is bound to the N-terminus of a glutamine synthetase monomer, a nucleic acid sequence encoding a fusion protein in which an HPLC6 peptide, which is a type I antifreeze protein, is bound to the N-terminus of a ferritin monomer, and the like. Other configurations

[0055] In addition to the configuration described above, the preparation kit of the present embodiment may also include a host cell, depending on the type of expression vector, a known buffer solution (such as phosphate-buffered saline (PBS)) for dissolving or dispersing the target protein, a film-hole grid generally used for a cryo-electron microscope sample, and the like.

[0056] Additionally, the cryo-electron microscope sample preparation kit according to the first embodiment and the cryo-electron microscope sample preparation kit according to the second embodiment can be used in combination as the preparation kit of the present embodiment.

[0057] That is, the preparation kit according to the first embodiment of the present invention can further contain a homomultimeric protein to which the antifreeze protein is bound.

[0058] Alternatively, the preparation kit according to the second embodiment of the present invention may further include an expression vector containing an antifreeze protein and a protein crosslinking agent, or a nucleic acid sequence encoding an antifreeze protein.

[0059] Accordingly, it is possible to solve three problems: distortion of the target protein's position relative to the electron beam incidence direction, localization of the target protein (e.g., adsorption to the amorphous carbon film), and control of the ice thickness in the cryo-electron microscope sample. That is, by using the cryo-electron microscope sample preparation kit according to the first embodiment and the cryo-electron microscope sample preparation kit according to the second embodiment in combination, a cryo-electron microscope sample can be obtained in which distortion of the target protein's position relative to the electron beam incidence direction is suppressed, the target protein is distributed over the entire observation area, and the ice thickness of the sample is uniformly controlled. Manufacturing process for a cryo-electron microscope sample

[0060] The manufacturing process for a cryo-electron microscope sample according to an embodiment of the present invention (hereinafter also referred to as the “manufacturing process of the present embodiment”) comprises manufacturing a cryo-electron microscope sample containing a target protein (hereinafter also referred to as the “preparation step”) using the cryo-electron microscope sample preparation kit according to the first embodiment or the second embodiment.

[0061] According to the manufacturing process of the present embodiment, it is possible, primarily using the cryo-electron microscope sample preparation kit of the first embodiment, to suppress distortion of the target protein's position in the sample with respect to the electron beam incidence direction. That is to say, the manufacturing process of the present embodiment can also be described as a method for controlling the position of a target protein in a cryo-electron microscope sample. The same applies if the preparation kit of the first embodiment further includes an ice thickness control molecule to which an amphipathic protein is bound.

[0062] According to the manufacturing process of the present embodiment, it is possible, primarily using the cryo-electron microscope sample preparation kit of the first embodiment, to prevent the target protein from colliding with or adsorbing onto the gas-liquid interface. That is to say, the manufacturing process of the present embodiment can also be described as a method for suppressing the adsorption of a target protein in a cryo-electron microscope sample onto a gas-liquid interface, or a method for suppressing the collision of a target protein in a cryo-electron microscope sample onto a gas-liquid interface. The same applies in a case where the preparation kit of the first embodiment further contains an ice thickness control molecule to which an amphipathic protein is bound.

[0063] According to the manufacturing process of the present embodiment, it is possible, primarily using the cryo-electron microscope sample preparation kit of the first embodiment, to suppress the adsorption of the target protein to the amorphous carbon film in the sample and to distribute the target protein over the entire observation area. That is to say, the manufacturing process of the present embodiment can also be described as a method for suppressing the adsorption of a target protein in a cryo-electron microscope sample to an amorphous carbon film. The same applies in a case where the preparation kit of the first embodiment further contains an ice thickness control molecule to which an amphipathic protein is bound.

[0064] According to the manufacturing process of the present embodiment, it is possible to control the ice thickness of the sample, primarily using the cryo-electron microscope sample preparation kit according to the second embodiment. That is to say, the manufacturing process of the present embodiment can also be described as a method for controlling the ice thickness in a cryo-electron microscope sample.

[0065] Next, the steps that constitute the manufacturing process of the present embodiment will be described in detail. Manufacturing step

[0066] In the manufacturing step, a cryo-electron microscope sample containing a target protein is prepared using the cryo-electron microscope sample preparation kit according to the first embodiment or the second embodiment.

[0067] Specifically, for example, in a case where the antifreeze protein is used as the preparation kit of the first embodiment, the antifreeze protein and the target protein are mixed in a buffer solution with a pH of 7 to 9.

[0068] In this case, the mixing ratio of the target protein and the antifreeze protein can be 1:1 to 1:5 or 1:1 to 1:2 with respect to the molar ratio. By adjusting the molar ratio within the range described above, it is possible to prevent the target protein from colliding with or adsorbing onto the gas-liquid interface. As a result, the distortion of the target protein's position relative to the electron beam incidence direction can be further suppressed, and the target protein can be projected in various orientations.

[0069] Additionally, for example, in a case where the protein crosslinking agent and the antifreeze protein are used in combination as the preparation kit of the first embodiment, the target protein and the antifreeze protein are first mixed in a solution in the presence of the protein crosslinking agent in order to bind the antifreeze protein to the target protein.

[0070] In this case, the mixing ratio of the target protein and the antifreeze protein can be 1:10 to 1:200 or 1:20 to 1:120 with respect to the molar ratio. By adjusting the molar ratio within the range described above, it is possible to bind the antifreeze protein sufficiently to the target protein. As a result, the distortion of the target protein's position relative to the electron beam incidence direction can be further suppressed, and the target protein can be projected in various positions.

[0071] Furthermore, the protein crosslinking agent can be used according to a known protocol, depending on its type. For example, in the case of an amine-reactive crosslinking agent, the agent is used at a concentration of approximately 10 times the molar amount or more and approximately 50 times the molar amount or less of the total molar amount of protein in a buffer solution with a pH of 7 to 9 that does not contain amines. The amine-reactive crosslinking agent is used in a solution with a specific concentration of approximately 0.25 mM to 15.00 mM. The crosslinking reaction is carried out, for example, for 1 minute or longer and 60 minutes or less at room temperature (approximately 25 °C) or for 1 hour or longer and 5 hours or less on ice. After the reaction, the crosslinking reaction is stopped by adding a solution containing a reaction terminator or by removing any unreacted amine-reactive crosslinking agent by dialysis or desalting.

[0072] Additionally, for example, in a case where an expression vector containing a nucleic acid sequence encoding an antifreeze protein is used as the preparation kit of the first embodiment, a gene encoding a target protein is inserted at a desired position in the expression vector. The insertion positions of the nucleic acid sequence encoding the antifreeze protein and the gene encoding the target protein in the expression vector are as described in the preparation kit of the first embodiment. Next, a fusion protein consisting of an antifreeze protein and a target molecule is expressed by transforming a suitable host cell with the expression vector containing a nucleic acid sequence encoding the antifreeze protein and a gene encoding the target molecule, according to the type of expression vector.The fusion protein is obtained from a culture solution of a host cell or from the interior of the host cell by lyse of the host cell. The resulting fusion protein can be purified as needed using a known method.

[0073] Alternatively, for example in a case where the preparation kit of the second embodiment is used, the homomultimeric protein to which the antifreeze protein is bound and the target protein are mixed in a buffer solution with a pH of 7 or higher and 9 or lower, which does not contain an amine. The target protein can be used as is, or a target protein to which the antifreeze protein is bound can be used with the preparation kit of the first embodiment.

[0074] The concentration of the homomultimeric protein to which the antifreeze protein is bound, and the concentration of the target protein in the mixed solution of the homomultimeric protein to which the antifreeze protein is bound, can be adjusted accordingly, depending on the type of homomultimeric protein being used. For example, in a case where the homomultimeric protein is ferritin, the concentration can be 0.1 µM or more and 3.0 µM or less, or 0.5 µM or more and 1.5 µM or less. Additionally, at this point, the concentration of the target protein in the mixed solution can be 1.0 µM or more and 5.0 µM or less, or 2.0 µM or more and 4.0 µM or less.In a case where the concentration of these proteins is within the range described above, the amount of homomultimeric protein to which the antifreeze protein is bound can be adjusted to an amount that does not interfere with the projection of the target protein, while uniform ice is formed according to the thickness or particle diameter of the homomultimeric protein.

[0075] Next, the solution containing the target protein to which the antifreeze protein is bound, or the solution containing the homomultimeric protein in which the target protein and the antifreeze protein are bound together, obtained as described above, is placed on the film-hole grid, which is then subjected to hydrophilization by glow discharge or similar method. After removing the excess solution using filter paper or similar, the grid is rapidly immersed in liquid ethane to freeze the sample. A frozen grid is thus obtained in which the target protein is encased in a thin amorphous ice film. The resulting frozen grid is stored in liquid nitrogen until it is observed using a cryo-electron microscope.

[0076] The cryo-electron microscope sample obtained by the manufacturing process of the present embodiment is mounted on a cryotransfer holder or a dedicated cartridge in a cryogenic workstation cooled with liquid nitrogen and is inserted into a cryo-electron microscope and imaged without freezing. It is possible to perform the structural analysis of the target protein by single-particle analysis based on the acquired data. Furthermore, the present invention can also be applied to structural analysis using subtomogram averaging with electron tomography.

[0077] Although each embodiment that uses the antifreeze protein (or the vector containing the nucleic acid sequence encoding the antifreeze protein) has been described above, the preparation kit of each of the embodiments described above may contain an amphipathic protein (or a vector containing a nucleic acid sequence encoding an amphipathic protein) different from the antifreeze protein, either instead of the antifreeze protein (or the vector containing the nucleic acid sequence encoding the antifreeze protein) or together with the antifreeze protein.

[0078] Additionally, in the manufacturing process of each of the embodiments described above, an amphipathic protein different from the antifreeze protein (or a vector containing a nucleic acid sequence encoding the amphipathic protein) can be used instead of the antifreeze protein (or the vector containing a nucleic acid sequence encoding the antifreeze protein) or together with the antifreeze protein.

[0079] The amphipathic protein has a portion that is highly hydrophobic and a portion that is highly hydrophilic. Since the hydrophobic portion of the amphipathic protein faces air, and the hydrophilic portion faces water, the amphipathic protein accordingly exhibits properties that cause it to accumulate at a gas-liquid interface, not just in the case of the antifreeze protein. Therefore, the same effect can also be achieved in a case where an amphipathic protein different from the antifreeze protein is used.

[0080] An amphipathic protein with a small and relatively simple structure is preferable, and an amphipathic protein with an amphipathic α-helix structure is particularly preferable. Examples of such a protein include a type I antifreeze protein, such as HPLC6, shown in examples, a KALA peptide, or the like, but the present invention is not limited thereto. Examples

[0081] The present invention is described below with reference to examples, but the present invention is not limited to the following examples. Example 1

[0082] Binding of antifreeze protein to target protein using protein crosslinking agents

[0083] By using a protein crosslinking agent to bind the antifreeze protein to an amino group on the surface of the target molecule, attempts were made to control the position of the target molecule in relation to the electron beam incidence direction. 1. Cross-linking reaction of HPLC6 and target protein

[0084] The target protein used was a hemagglutinin (HA) protein (manufactured by MyBioSource, Inc., MBS434205). The antifreeze protein used was an HPLC6 peptide (amino acid sequence: SEQ ID NO: 1), a type I antifreeze protein, expressed in Escherichia coli as a GST fusion protein, and then GST was cleaved and purified for use.The HA protein (concentration: 1 mg / ml) and the HPLC6 peptide (concentration: 1.7 mg / ml), each dissolved in PBS, were mixed in a molar ratio of 1:0, 1:4, 1:22 or 1:110. The mixture was subjected to a crosslinking reaction at room temperature (about 25 °C) for 5 minutes in the presence of 0.14 wt% glutaraldehyde as a protein crosslinking agent. The crosslinking reaction was stopped by adding a 1 M Tris hydrochloride buffer solution (pH 8.0) so that a final concentration of 44 mM was achieved. Then each sample solution (concentration of HA protein: 1.6 µM (the concentration of HA protein is the same in each sample, and the concentration of HPLC6 peptide is different)) was prepared. 2. Preparation of the sample grid

[0085] After hydrophilic treatment of a film-perforated grid (Quantifoil R1.2 / 1.3 Cu 300) by glow discharge, 3 µl of the sample solution prepared in step 1 was placed on the grid. Next, the excess solution was removed with filter paper, and then the grid was rapidly immersed in liquid ethane to freeze the sample. This resulted in a frozen grid in which the protein sample was encased in a thin amorphous ice film. The resulting frozen grid was stored in liquid nitrogen until it was observed using a cryo-electron microscope. 3. Observation with a cryo-electron microscope

[0086] Each of the frozen grids produced in section "2." was placed in a cryo-electron microscope (manufactured by Thermo Fisher Scientific, Inc., Talos Arctica) and observed and imaged at an accelerating voltage of 200 kV. Using RELION software, particle images were extracted from the acquired image, projected in different orientations, classified into particle images in the same orientation, aligned, and then the orientations and positions of the classified particle images for each projection direction were aligned to obtain a mean image. The results are presented in Fig. 1 shown. In Fig. 1 is the total number of projected particle images, as described next to the molar ratio of the HA protein to HPLC6. Furthermore, the number described on the particle image indicates the number of particle images in the same orientation, and the particle images were aligned from left to right in order of the number of particle images. Additionally, in Fig. 1 The reference symbol “T” indicates a particle image obtained by projecting the HA protein from above, and the reference symbol “S” indicates a particle image obtained by projecting the HA protein from the side.

[0087] As in Fig. As shown in Figure 1, the number of side-projected particle images of the HA protein tends to increase with increasing molar ratio of the HA protein to the HPLC6 peptide. In a case where the molar ratio of the HA protein to the HPLC6 peptide is 1:0 to 1:22, the number of top-projected particle images of the HA protein is greatest, but in a case where the molar ratio of the HA protein to the HPLC6 peptide is 1:110, the number of side-projected particle images of the HA protein is greatest.It is assumed that, as the molar fraction of HPLC6 peptide increases, the amount of HPLC6 peptide bound to the lysine residue or the N-terminal amino group present on the side (side surface) of the HA protein increases, and the surface (side surface) to which the HPLC6 peptide is bound faces the gas-liquid interface, the side-projected particle image of the HA protein increases.

[0088] Therefore, it was confirmed that the position of the target protein relative to the electron beam incidence direction can be controlled by increasing the molar ratio of the target protein to the HPLC6 peptide, thereby enabling the collection of a large number of particle images projected in different directions. Example 2: Effect of the suppression of the adsorption of a target protein to an amorphous carbon film by antifreeze protein

[0089] It was investigated that for a target protein with the property of readily adsorbing onto an amorphous carbon film in a lattice, a fusion protein from an antifreeze protein and a target protein is genetically engineered, thereby suppressing adsorption onto the amorphous carbon film and distributing the target protein over the entire observation area. 1. Expression and purification of a fusion protein

[0090] Glutamine synthetase was used as the target protein. Glutamine synthetase consists of a homododecamer and has a hexagonal columnar shape with a diameter of approximately 16 nm and a height (thickness) of approximately 11 nm. The glutamine synthetase monomer self-assembles in solution to form a dodecamer. A nucleic acid sequence encoding a fusion protein, specifically an antifreeze protein, in which an HPLC6 peptide (amino acid sequence: SEQ ID NO: 1), a type I antifreeze protein, is linked via a linker sequence (amino acid sequence: SEQ ID NO: 4, base sequence: SEQ ID NO: 5) to the N-terminus of a glutamine synthetase monomer (amino acid sequence: SEQ ID NO: 3), was inserted into the plasmid vector pET-28a (manufactured by Novagen Inc.). A strain of Escherichia coli BL21 (DE3) was transformed using the vector.The target protein was purified from a soluble fraction of induced Escherichia coli using a nickel column, and after cleavage of the His tag, the target protein was further purified using a gel filtration column.

[0091] Additionally, as a control, a nucleic acid sequence (base sequence: SEQ ID NO: 6) encoding only one glutamine synthetase monomer to which no antifreeze protein is bound was inserted into a plasmid vector, and expression and purification were performed in the same way using an Escherichia coli expression system. 2. Preparation of the sample grid

[0092] Sample grid preparation: For samples containing glutamine synthetase bound to the HPLC6 peptide, the protein concentration was adjusted to 2.0 µM. For samples containing only glutamine synthetase, the protein concentration was adjusted to 2.0 µM. The samples were diluted with a buffer solution (composition: 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl₂) to achieve a protein concentration of 2.0 µM. The sample grids were prepared using the same procedure as described in "2." of Example 1. 3. Observation with a cryo-electron microscope

[0093] Each of the frozen grids produced in section "2." was placed in a cryo-electron microscope (manufactured by Thermo Fisher Scientific, Inc., Talos Arctica) and observed and imaged at an accelerating voltage of 200 kV. The results are presented in Fig. 2A (glutamine synthetase only) and Fig. 2B (glutamine synthetase to which HPLC6 peptide is bound) is shown.

[0094] As in Fig. As shown in Figure 2A, in the sample grid containing only glutamine synthetase, glutamine synthetase was not observed in the center of the hole, and glutamine synthetase was adsorbed onto the amorphous carbon film in the periphery of the hole and was not distributed throughout the entire observation area.

[0095] On the other hand, as in Fig. 2B shows that in the sample grid containing the glutamine synthetase to which the HPLC6 peptide was bound, particles were evenly distributed both in the center of the hole and in the periphery of the hole.

[0096] Furthermore, in Fig. 2B predominantly observed a particle image obtained by projecting a dodecamer of glutamine synthetase from above. It was assumed that in the sample, the glutamine synthetase monomer to which the HPLC6 peptide was bound at the N-terminus formed a dodecamer, and that the surface (top surface) to which the HPLC6 peptide was bound faced the gas-liquid interface, thus aligning the position of the glutamine synthetase dodecamer with respect to the electron beam incidence direction, resulting in a predominantly top-projected particle image.

[0097] Furthermore, the overall image of the hole confirmed that the interior of the hole was whitish overall and that a uniform and thin layer of ice had formed.

[0098] Therefore, it was confirmed that for a target protein that is readily adsorbed onto the amorphous carbon film in the lattice, fusion with an antifreeze protein suppresses adsorption onto the amorphous carbon film and allows particles of the target protein to be distributed across the entire observation area.

[0099] It has also been shown that by using homomultimeric proteins with uniform thickness and particle size, the ice thickness on the grid can be kept thin and uniform. Example 3: Control of ice thickness by homomultimeric protein to which antifreeze protein is bound

[0100] The study investigated whether the ice thickness on the grid could be kept thin and uniform by using homomultimeric proteins other than glutamine synthetase, which have a uniform thickness and particle size, as fusion proteins bound to antifreeze proteins. 1. Expression and purification of a fusion protein

[0101] Ferritin was used as the target protein. In Example 3, ferritin serves as both the target molecule and a molecule for controlling ice thickness. Ferritin consists of a homo-24 mer and has a spherical shape with a diameter of approximately 14 nm. The ferritin monomer self-assembles in solution to form a 24-mer. A nucleic acid sequence encoding a fusion protein that acts as an antifreeze protein, in which an HPLC6 peptide (amino acid sequence: SEQ ID NO: 1), a type I antifreeze protein, is linked via a linker sequence (amino acid sequence: SEQ ID NO: 8, base sequence: SEQ ID NO: 9) to the N-terminus of a ferritin monomer (amino acid sequence: SEQ ID NO: 7), was inserted into the plasmid vector pET-28a (manufactured by Novagen Inc.). Escherichia coli was transformed with the vector.The target protein was purified from a soluble fraction of induced Escherichia coli using a nickel column, and after cleavage of the His tag, the target protein was further purified using a gel filtration column.

[0102] Additionally, as a control, a nucleic acid sequence (base sequence: SEQ ID NO: 10) encoding only one ferritin monomer to which no antifreeze protein is bound was inserted into a plasmid vector, and expression and purification were performed in the same way using an Escherichia coli expression system. 2. Preparation of the sample grid

[0103] For the sample containing the ferritin to which the HPLC6 peptide was bound, the sample was diluted with a buffer solution (composition: 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2) to achieve a protein concentration of 2 µM, and for the sample containing only ferritin, the sample was diluted with the buffer solution to achieve a protein concentration of 7 µM, and the sample grid was prepared using the same procedure as in “2.” of Example 1. 3. Observation with a cryo-electron microscope

[0104] Each of the frozen grids produced in section "2." was placed in a cryo-electron microscope (manufactured by Thermo Fisher Scientific, Inc., Talos Arctica) and observed and imaged at an accelerating voltage of 200 kV. The results are presented in Fig. 3 shown (left: ferritin only, right: ferritin-HPLC6 fusion peptide).

[0105] As in Fig. Figure 3 shows that in the sample grid containing only ferritin, only the center of the hole was whitish, and ice was only thin in the middle part.

[0106] On the other hand, it was confirmed that in the sample lattice containing the ferritin-HPLC6 fusion peptide, the interior of the hole was whitish overall and a uniform and thin layer of ice had formed. It was assumed that in the sample, the ferritin monomers to which the HPLC6 peptide was bound at the N-terminus formed a 24-mer structure, and that the surface (top surface) to which the HPLC6 peptide was bound faced the gas-liquid interface and was distributed across the entire hole, resulting in a uniform ice thickness corresponding to the diameter of the ferritin.

[0107] Therefore, it was confirmed that, regardless of the type of homomultimeric protein, the ice thickness on the grid can be kept thin and uniform by using homomultimeric proteins with uniform thickness and particle size. Example 4: Control of ice thickness and the dispersion effect of the target protein by a homomultimeric protein to which an antifreeze protein is bound.

[0108] It was investigated that the target protein, which has the property of readily accumulating in the middle region of the observation area of ​​the grid, was mixed with the homomultimeric protein to which the antifreeze protein was bound, thereby keeping the accumulation of the target protein in the middle region of the observation area of ​​the grid under control, while keeping the ice thickness of the grid thin and uniform and distributing the target protein over the entire observation area. 1. Expression and purification of a fusion protein

[0109] The target protein used was an HA protein (manufactured by MyBioSource, Inc., MBS434205). The fusion protein used was a ferritin-HPLC6 fusion protein, a type I antifreeze protein, in which the HPLC6 peptide, a type I antifreeze protein, was bound to the N-terminus of the ferritin monomer and was expressed and purified using the same procedure as in "1." of Example 3 described above. 2. Preparation of the sample grid

[0110] For a sample containing the HA protein and the ferritin-HPLC6 fusion peptide, the sample was diluted with a buffer solution (composition: 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2) such that the concentration of the HA protein was 3.0 µM and the concentration of the ferritin-HPLC6 fusion peptide was 1.0 µM, and the sample grid was prepared using the same procedure as in “2.” in Example 1. 3. Observation with a cryo-electron microscope

[0111] Each of the frozen grids produced in section "2." was placed in a cryo-electron microscope (manufactured by Thermo Fisher Scientific, Inc., Talos Arctica) and observed and imaged at an accelerating voltage of 200 kV. The results are presented in Fig. 4 shown.

[0112] As shown in the picture on the left side of Fig. As shown in Figure 4, it was confirmed that the interior of the hole was whitish overall and that a uniform and thin layer of ice had formed. Additionally, as shown in the image on the right of Figure 4, Fig. Figure 4 shows the HA protein distributed throughout the entire observation area.

[0113] Therefore, it was confirmed that by using homomultimeric proteins to which the antifreeze protein is bound, the ice thickness on the grid can be kept thin and uniform through fusion with an antifreeze protein. Example 5

[0114] Suppression of positional distortion by suppressing the adsorption of the target protein to a gas-liquid interface by protecting the gas-liquid interface with antifreeze protein.

[0115] For a target protein that tends to adsorb in a specific position at the gas-liquid interface, it was investigated whether the distortion of the target protein's position could be suppressed by protecting the gas-liquid interface through the addition of an antifreeze protein and preventing the target protein from colliding with and adsorbing the gas-liquid interface. 1. Production of a target protein and an antifreeze protein

[0116] The target molecule used was the inosine 5'-monophosphate dehydrogenase 2 (IMPDH2) mutant Y12A (hereafter sometimes referred to as "IMPDH2 (Y12A)"). IMPDH2 (Y12A) was generated by introducing the Y12A mutation into a plasmid containing the gene for wild-type IMPDH2 and subsequently expressing it in E. coli. The type I antifreeze protein HPLC6 peptide (amino acid sequence: SEQ ID NO: 1), expressed in E. coli as a GST fusion protein, was used as the antifreeze protein, after which the GST was cleaved and purified. 2. Preparation of the sample grid

[0117] For the sample containing IMPDH2 (Y12A) and HPLC6 peptide, the sample was diluted with a buffer solution (composition: 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2) to achieve a molar ratio of IMPDH2 (Y12A) to HPLC6 peptide of 1:1.25, and the sample grid was prepared using the same procedure as described in "2." in Example 1. A film-hole grid (Quantifoil R1.2 / 1.3 Cu 300) and a graphene grid (manufactured by AirMembrane Co., Ltd., 2-layer graphene TEM grid Quantifoil R1.2 / 1.3 300 mesh Au hydrophilized) were used. A control sample containing only the target molecule was prepared. 3. Observation with a cryo-electron microscope

[0118] Each of the frozen grids produced in section "2." was placed in a cryo-electron microscope (manufactured by Thermo Fisher Scientific, Inc., Talos Arctica) and observed and imaged at an accelerating voltage of 200 kV. The results are presented in Fig. 5 shown. In Fig. 5 The reference symbol “T” indicates a particle image obtained by projecting IMPDH2 (Y12A) from above, and the reference symbol “S” indicates a particle image obtained by projecting IMPDH2 (Y12A) from the side.

[0119] As in (A) and (C) of Fig. As shown in Figure 5, in a sample containing only IMPDH2 (Y12A), which is a target protein, IMPDH2 (Y12A) was adsorbed onto a gas-liquid interface in a specific position, and the particle images of IMPDH2 (Y12A) projected from above constituted the majority of the images. On the other hand, as shown in Figures (B) and (D) of Fig. Figure 5 shows that in the sample containing IMPDH2 (Y12A) and the HPLC6 peptide, a particle image was also observed in which IMPDH2 (Y12A) was projected from the side or from an oblique angle above.

[0120] It was therefore confirmed that in a case where the target protein and the HPLC6 peptide were mixed and used, the target protein was prevented from colliding with and adsorbing onto the gas-liquid interface, and a large number of particle images projected in different directions could be collected. Example 6

[0121] Effect of suppressing the adsorption of a target protein to an amorphous carbon film by another amphipathic protein as an antifreeze protein. For target proteins that tend to be adsorbed onto the amorphous carbon film in the lattice, the possibility of inhibiting adsorption onto the amorphous carbon film and dispersing the target protein throughout the observation area was investigated by creating a fusion protein from the target protein with another amphipathic protein as an antifreeze protein. 1. Expression and purification of the fusion protein

[0122] Ferritin was used as the target protein. A nucleic acid sequence encoding a fusion protein, in which a protein consisting of an amino acid sequence (SEQ ID NO: 11) containing a KALA peptide (an example of an amphipathic protein that is not an antifreeze protein) was linked to the N-terminus of a ferritin monomer, was inserted into the plasmid vector pET-28a (manufactured by Novagen Inc.). In this fusion protein, a ferritin monomer and a protein containing a KALA peptide are linked via a linker sequence (amino acid sequence: SEQ ID NO: 8). A strain of Escherichia coli BL21 (DE3) was transformed with the vector. The target protein was purified from a soluble fraction of the induced Escherichia coli using a nickel column, and after His-tag cleavage, the target protein was further purified using a gel filtration column.

[0123] Additionally, a control sample containing only ferritin was also prepared using the Escherichia coli expression system without the sequence of the amphipathic protein, as described in more detail under “1.” of Example 3. 2. Preparation of the sample grid

[0124] For the sample containing ferritin to which a KALA peptide was bound, and the sample containing only ferritin, the samples were each diluted with a buffer solution (composition: 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2) to a protein concentration of 2.0 µM, and the frozen lattice of the sample was prepared using the same procedure as in “2.” of Example 1. 3. Observation with a cryo-electron microscope

[0125] Each of the frozen grids produced in section “2.” was placed in a cryo-electron microscope (manufactured by Thermo Fisher Scientific, Inc., Talos Arctica) and observed and imaged at an accelerating voltage of 200 kV.

[0126] Fig. 6A is an image of ferritin taken with a cryo-electron microscope in Example 6, and Fig. 6B is an image of a fusion protein of ferritin and a KALA peptide, taken with a cryo-electron microscope in Example 6.

[0127] As in Fig. As shown in Figure 6A, in the frozen lattice containing only ferritin, the ferritin was not observed in the center of the hole, the ferritin was adsorbed onto the amorphous carbon film in the periphery of the hole, and the ferritin was not distributed over the entire observation area.

[0128] In contrast, as in Fig.Figure 6B shows that in the frozen lattice containing the ferritin to which the KALA peptide was bound, the particles of the fusion protein are evenly distributed both in the center of the hole, indicated by the arrow, and in the periphery of the hole.

[0129] The above results confirmed that even in the case of a different amphipathic protein, other than an antifreeze protein, the adsorption of the target protein to the amorphous carbon film at the periphery of the hole can be effectively suppressed. It is believed that the present embodiment can be applied to proteins other than HPLC6, for example, a protein with a different amphipathic α-helix structure, other antifreeze proteins, and other amphipathic proteins. Commercial applicability

[0130] According to the cryo-electron microscope sample of the present embodiment, it is possible to obtain a cryo-electron microscope sample in which distortion of an undesired position of the target protein in a sample with respect to an electron beam incidence direction and collision with and adsorption at a gas-liquid interface are suppressed, and in which the target protein is distributed over the entire observation area. Furthermore, according to the cryo-electron microscope sample of the present embodiment, it is possible to obtain a cryo-electron microscope sample in which the ice thickness of a sample is uniformly controlled. Reference symbol list S particle image obtained by projecting protein from the side T particle image obtained by projecting protein from above QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited non-patent literature

[0000] 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

[0008]

Claims

[1] Cryo-electron microscope sample preparation kit, comprising: an amphipathic protein or an expression vector that includes a nucleic acid sequence encoding the amphipathic protein. [2] Cryo-electron microscope sample preparation kit, including: an ice thickness control molecule to which an amphipathic protein is bound. [3] Cryo-electron microscope sample preparation kit according to claim 1 or 2, wherein the amphipathic protein has an amphipathic α-helix structure. [4] Cryo-electron microscope sample preparation kit according to claim 1 or 2, wherein the amphipathic protein is an antifreeze protein. [5] Cryo-electron microscope sample preparation kit according to claim 4, wherein the antifreeze protein is a type I antifreeze protein. [6] Cryo-electron microscope sample preparation kit according to claim 5, wherein the type I antifreeze protein is an HPLC6 peptide. [7] Cryo-electron microscope sample preparation kit according to claim 1 or 2, wherein the amphipathic protein is a KALA peptide. [8] Cryo-electron microscope sample preparation kit according to claim 1, comprising: a protein crosslinking agent. [9] Cryo-electron microscope sample preparation kit according to claim 8, wherein the protein crosslinking agent is an amine-reactive crosslinking agent. [10] Cryo-electron microscope sample preparation kit according to claim 9, wherein the amine-reactive crosslinking agent is glutaraldehyde or bis(3-sulfo-N-succinimidyl)suberate or a salt thereof. [11] Cryo-electron microscope sample preparation kit according to claim 1, comprising: an ice thickness control molecule to which an amphipathic protein is bound. [12] Cryo-electron microscope sample preparation kit according to claim 2 or 11, wherein the ice thickness control molecule is an antifreeze protein and a homomultimeric protein. [13] Cryo-electron microscope sample preparation kit according to claim 12, wherein the homomultimeric protein is a glutamine synthetase or ferritin. [14] Method for producing a cryo-electron microscope sample, comprising: Preparation of a cryo-electron microscope sample containing a target protein using the cryo-electron microscope sample preparation kit according to claim 1 or 2. [15] Method for controlling the position of a target protein in a cryo-electron microscope sample, the method comprising: Preparation of a cryo-electron microscope sample containing a target protein using the cryo-electron microscope sample preparation kit according to claims 1, 8, or 11. [16] Method for suppressing the adsorption of a target protein in a cryo-electron microscope sample onto an amorphous carbon film, the method comprising: Preparation of a cryo-electron microscope sample containing a target protein using the cryo-electron microscope sample preparation kit according to claims 1, 8 or 11. [17] Method for suppressing the collision and adsorption of a target protein in a cryo-electron microscope sample onto a gas-liquid interface, the method comprising: Preparation of a cryo-electron microscope sample containing a target protein using the cryo-electron microscope sample preparation kit according to claims 1, 8 or 11. [18] Method for controlling ice thickness in a cryo-electron microscope sample, the method comprising: Preparation of a cryo-electron microscope sample containing a target protein using the cryo-electron microscope sample preparation kit according to claim 2, or 11.

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