Affinity carrier net for cryoelectron microscope and preparation method and application of frozen sample of affinity carrier net

Abstract

The invention provides an affinity carrier net for a cryoelectron microscope and a preparation method and application of a frozen sample of the affinity carrier net. The affinity carrier net comprises a carbonaceous bearing membrane covering the carrier net, a small organic molecule pyr-NHS connected with the carbonaceous bearing membrane, and a specific antibody protein connected with the small organic molecule pyr-NHS; the carbonaceous bearing film is a graphene film, a graphene oxide film or a carbon film. The method comprises the following steps: performing fusion expression on a PA tag having strong affinity interaction with an NZ-1 antibody on a target protein, and performing mixed incubation on an affinity carrier net fixed with an NZ-1 antibody protein and a cell lysis solution of the target protein which performs fusion expression on the PA tag; according to the method, efficient affinity purification and direct cryoelectron microscope sample preparation of the target protein with the PA tag in the cell lysis solution are realized, data collection and high-resolution structural analysis are carried out, the sample purification time and steps are reduced, the sample integrity is improved, the sample demand is reduced, and meanwhile, the problem of orientation tendency of sample distribution is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of cryo-electron microscopy, and in particular to a preparation method and application of an affinity grid for cryo-electron microscopy and frozen samples thereof. Background Art

[0002] Since its emergence in the 1970s, cryo-electron microscopy (cryo-EM) has undergone a long period of development, especially based on the technological innovation of hardware and software in the past decade or so. It has become the most important means of analyzing the near-atomic resolution structure of biological macromolecules, which has greatly promoted the research process of clarifying biological mechanisms at the molecular level. First, the biological sample is dripped onto a grid covered by a support film, and then quickly frozen by liquid ethane or propane cooled by liquid nitrogen to form a glassy amorphous ice layer, so that the biological macromolecules remain in a near physiological state in the frozen sample, and the embedding of a certain thickness of glassy ice also reduces the radiation damage of the electron beam to the biological macromolecules. Finally, the three-dimensional structure of the biological macromolecule can be obtained by collecting data and reconstructing calculations on the frozen sample using a transmission electron microscope.

[0003] With the development of electron microscopy technology, electron microscopes, detectors, and image processing algorithms are no longer the problems that restrict structural analysis. However, cryo-EM technology still faces some bottlenecks, which seriously limit the efficiency of this technology in analyzing the structure of biological molecules. One of the important bottlenecks is the preparation of frozen samples, which usually includes a series of tedious sample purification processes and the preparation of frozen grids for electron microscopy observation. At present, the main problems in the preparation of frozen samples include the difficulty in obtaining samples of biological macromolecules and super-macromolecule complexes, the damage to biological samples caused by the gas-liquid interface during the sample preparation process, the orientation tendency of the distribution of biological samples, and the extremely cumbersome purification process of biological samples in the early stage of cryo-EM sample preparation. These problems have led to the need for many structural biology studies to invest a lot of time and energy in sample optimization. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an affinity grid for cryo-electron microscopy and a method for preparing frozen samples thereof and an application thereof. The affinity grid for cryo-electron microscopy provided by the present invention and a method for preparing frozen samples of a target protein in a direct affinity cell lysate and an application thereof can effectively reduce the time and steps of sample purification, improve sample integrity, reduce sample demand, and overcome the problems of sample fragility and sample distribution orientation tendency caused by the gas-liquid interface. At the same time, the present invention provides a method in which the affinity grid is directly used for a target protein with a PA tag in an affinity cell lysate to prepare a frozen sample.

[0005] In the first aspect, the present invention provides an affinity grid for cryo-electron microscopy, comprising a carbonaceous support film covering the grid, an organic small molecule pyr-NHS connected to the carbonaceous support film, and a specific antibody protein connected to the organic small molecule pyr-NHS; the carbonaceous support film is a graphene film, a graphene oxide film or a carbon film. In the present invention, the organic small molecule pyr-NHS with a conjugated structure interacts with the carbon atoms covered on the surface of the grid through π-π stacking, and the hybridization state is sp 2 Hybridized graphene, graphene oxide or carbon film is closely combined with the above sp 2 The surface of the hybridized specific carbonaceous carrier film is modified by non-covalent bonds, and at the same time, the N-hydroxysuccinimide ester active group of pyr-NHS undergoes a cross-linking reaction with the lysine side chain of the antibody protein and the primary amine at the amino end, and the antibody NZ-1 is fixed on the carrier surface. The affinity carrier obtained by the present invention can directly express the target protein of the PA tag from the cell lysate for affinity fusion to perform freezing sample preparation and high-resolution cryo-electron microscopy structure analysis, which simplifies the protein purification process and is easy to operate. It can be applied to antigen-antibody and other affinity-based reaction systems to achieve anchoring of different proteins. The affinity carrier provided by the present invention is applied to freezing sample preparation for cryo-electron microscopy research, and can realize the specific enrichment of the target protein directly from the cell lysate by the affinity carrier, which greatly simplifies the purification process of the protein complex before the preparation of the frozen sample. In addition, there is no need to treat the carbonaceous carrier film with oxygen plasma, nor is there a need to treat the carrier with a buffer of special components. The specific affinity grid provided by the present invention can better anchor the target protein on the grid surface, especially overcomes the problems of cumbersome protein purification process, protein fragility caused by gas-liquid interface, distribution orientation tendency, etc. The present invention improves the success rate of freezing sample preparation while improving the quality of frozen samples prepared by the affinity grid, and improves the efficiency of sample preparation. The high-resolution structure of the complex can be reconstructed through the conventional single particle analysis process. The frozen sample prepared by affinity target protein directly from cell lysate using the affinity grid has a clean background and can be better used for subsequent cryo-electron microscopy data collection and high-resolution structure analysis.

[0006] Preferably, the specific antibody protein is NZ-1 antibody protein.

[0007] Further preferably, the affinity support grid is prepared by a method comprising the following steps: using an organic small molecule pyr-NHS to immobilize the NZ-1 antibody protein on the surface of the carbonaceous support film covering the support grid.

[0008] In a second aspect, the present invention provides a method for preparing an affinity grid for cryo-electron microscopy, comprising the following steps: using an organic small molecule pyr-NHS to fix a specific antibody protein on the surface of a carbonaceous carrier film covering the grid to obtain an affinity grid; the carbonaceous carrier film is a graphene film, a graphene oxide film or a carbon film; the specific antibody protein is preferably an NZ-1 antibody protein.

[0009] Preferably, the method for preparing the affinity grid for cryo-electron microscopy comprises:

[0010] 1) immersing a grid covered with a graphene film, a graphene oxide film or a carbon film in a pyr-NHS solution for reaction;

[0011] 2) cleaning and drying the carrier mesh treated in step 1) to obtain a carrier mesh with pyr-NHS attached to the surface;

[0012] 3) Using the pyr-NHS on the surface of the grid obtained in step 2) as a cross-linking agent, the NZ-1 antibody solution is coated on the surface of the grid and incubated to obtain an affinity grid.

[0013] Further preferably, in step 1), the concentration of the pyr-NHS solution is 0.8-1.2 mM; the immersion temperature is 10-30° C., and the immersion time is 45 min-2 h; preferably, the step comprises dissolving pyr-NHS in a first organic solvent to obtain the pyr-NHS solution; and the first organic solvent is preferably DMF or methanol.

[0014] Further preferably, in step 2), the cleaning step comprises: dripping a second organic solvent on the carrier mesh, and then absorbing the surface liquid from the side edge of the carrier mesh, and the cleaning step is repeated 3 to 5 times; preferably, the second organic solvent comprises DMF or methanol, and 5 to 10 μL is dripped each time; the drying temperature is preferably 16 to 26°C, and the drying time is 10 to 15 min.

[0015] Further preferably, step 3) comprises dropping 5 to 10 μL of a NZ-1 antibody solution having a final concentration of 25 to 100 μg / mL onto the surface of the grid and incubating at 4 to 25°C for 45 min to 3 h to obtain an affinity grid having the NZ-1 antibody protein fixed on the surface of the grid; preferably, the solvent of the NZ-1 antibody solution is a buffer solution containing no amino groups, preferably PBS buffer, acetate buffer or HEPES buffer; after the incubation, preferably, the step of washing the surface of the grid with a second buffer solution is further included; the second buffer solution is a buffer solution containing amino groups, preferably Tris buffer or glycine solution.

[0016] In a third aspect, the present invention also provides the use of the affinity grid for cryo-electron microscopy or the affinity grid obtained by the preparation method of the affinity grid for cryo-electron microscopy in preparing frozen samples for cryo-electron microscopy; preferably, the affinity grid is used to directly and specifically enrich the target protein containing the PA tag from the cell lysate.

[0017] In a fourth aspect, the present invention provides a method for preparing a frozen sample, comprising: using an organic small molecule pyr-NHS to fix NZ-1 antibody protein on the surface of a carbonaceous carrier film covering the carrier network to prepare an affinity carrier network; incubating the affinity carrier network with a cell lysate containing a target protein, so that the affinity carrier network has an affinity for the target protein from the cell lysate, and directly freezing the sample; the target protein in the cell lysate is a target protein fused with an expression PA tag, and the carbonaceous carrier film is a graphene film, an oxidized graphene film or a carbon film.

[0018] Preferably, the method for preparing frozen samples comprises subjecting a cell lysate to high-speed centrifugation, ultracentrifugation and glycerol density gradient centrifugation to obtain a crude cell lysis solution containing a target protein fused with a PA tag; fixing the affinity grid in a cell culture plate, and adding the crude cell lysis solution to the cell culture plate for incubation.

[0019] Further preferably, the method for preparing the frozen sample comprises centrifuging the cell lysate at 10,000 to 50,000 g for 15 to 45 min, then ultracentrifuging at 100,000 to 200,000 g for 1 to 3 h, and then performing glycerol density gradient centrifugation; placing the affinity grid in a cell culture plate, adding a crude cell lysis solution containing the target protein until the affinity grid is covered; incubating at 4 to 25° C. for 20 to 60 min, preferably, using a pipette to mix the crude cell lysis solution every 5 to 10 min to allow the affinity grid to bind to the target protein.

[0020] In a fifth aspect, the present invention provides an application of the frozen sample obtained by the frozen sample preparation method in the analysis of biological macromolecule structure, wherein the application is any of the following:

[0021] 1) Avoid damage to biomacromolecules caused by the gas-liquid interface during sample preparation;

[0022] 2) Overcoming the orientation tendency problem of biological sample distribution;

[0023] 3) Achieve affinity of target protein directly from cell lysate for frozen sample preparation;

[0024] 4) Collect cryo-EM data and conduct high-resolution cryo-EM structure analysis.

[0025] According to a preferred embodiment of the present invention, the high-affinity PA-NZ-1 affinity system is used for assembly in the present invention to verify that the affinity network of the present invention has the best application effect. However, the affinity system applicable to the present invention includes but is not limited to the PA-NZ-1 affinity system, and can also be applied to other protein interaction systems with high affinity, such as the FLAG-M2 affinity system, the HA-HA tag antibody affinity system, etc.

[0026] The beneficial effect of the present invention is at least that: the present invention utilizes the organic small molecule pyr-NHS with a conjugated structure to form a hybrid state of sp with the carbon atoms covered on the surface of the carrier through π-π stacking interaction. 2 The hybridized graphene, graphene oxide or carbon film is tightly combined, and its surface is non-covalently modified. At the same time, the N-hydroxysuccinimide ester active group of pyr-NHS undergoes a cross-linking reaction with the lysine side chain of the antibody protein and the primary amine at the amino end, and the NZ-1 antibody is fixed on the surface of the grid, thereby obtaining an affinity grid. The affinity grid fixed with the NZ-1 antibody can then be directly used for the target protein with a PA tag that is specifically recognized by the affinity antibody from the cell lysate, and frozen sample preparation is performed for the study of high-resolution structural analysis of cryo-electron microscopy. The preparation of the affinity grid provided by the present invention and the method for applying the affinity grid to frozen sample preparation have a simple process and are easy to operate, which greatly simplifies the cumbersome protein purification process in the early stage of cryo-electron microscopy sample preparation. The affinity grid provided by the present invention can anchor the target protein to the surface of the grid through antibodies, which overcomes the problems of protein fragility and distribution orientation tendency caused by the gas-liquid interface to a certain extent, and improves the success rate of frozen sample preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 A schematic diagram of the preparation of the affinity grid provided in Example 1 of the present invention and its application in specific affinity target protein in cell lysate and freezing sample preparation;

[0029] Figure 2Characterization of the surface properties of the affinity grid provided in Example 1 of the present invention; wherein (A) is an X-ray photoelectron energy spectrum of the surface N1s element of the graphene oxide grid before (left) and after (right) treatment with pyr-NHS; (B) is a 120kV transmission electron microscope photo of the negatively stained samples of the graphene oxide grid (left) and the affinity grid (right); (C) is an atomic force microscope three-dimensional height morphology of the graphene oxide grid (left) and the affinity grid (right); (D) is a 120kV transmission electron microscope photo of the negatively stained samples of the graphene oxide grid covered with pyr-NHS treated with different concentrations of NZ-1Fab;

[0030] Figure 3 The schematic diagram of the target protein sequence and the purity characterization of the crude cell lysis solution provided in Example 2 of the present invention; wherein (A) is a schematic diagram of the sequence of the target protein; Figure 3 (B) is the SDS-PAGE electrophoresis Coomassie Brilliant Blue staining of the crude cell lysate of TBCA-apoferritin with PA label; (C) is the 120 kV transmission electron microscopy photo of the negatively stained sample of the crude cell lysate of TBCA-apoferritin with PA label;

[0031] Figure 4 A schematic diagram of the process of preparing the affinity grid provided in Example 2 of the present invention and applying it to directly affinity target protein in cell lysate and freezing sample preparation;

[0032] Figure 5 A 200 kV cryo-electron micrograph of the affinity grid provided in Example 2 of the present invention applied to direct affinity target protein in cell lysate; wherein (A) is a 200 kV cryo-electron micrograph of a graphene oxide supported membrane grid (left) and a graphene oxide supported membrane affinity grid (right) incubated with a cell lysate to prepare a sample; (B) is a 200 kV cryo-electron micrograph of a continuous carbon film grid (left) and a continuous carbon film affinity grid (right) incubated with a cell lysate to prepare a sample;

[0033] Figure 6 The affinity grid frozen sample provided in Example 3 of the present invention is applied to high-resolution structural research; wherein (A) is a three-dimensional reconstructed density map of the TBCA-apoferritin complex in the cell lysate using the affinity grid for direct affinity; (B) is a high-resolution density map of the representative amino acid side chains of apoferritin in TBCA-apoferritin; (C) is a TBCA density display map in the TBCA-apoferritin reconstruction results using ordinary graphene oxide grids (left) and affinity grid frozen samples (right);

[0034] Figure 7The application of the affinity carrier provided in Example 4 of the present invention to overcome the problem of the dominant orientation of protein samples; wherein Figure 7 (A) Cryo-EM images of yeast CCT6 subunit homo-oligomeric ring complex prepared using common holey carbon R2 / 1 copper mesh and the average results of two-dimensional classification; Figure 7 (B) Cryo-electron microscopy image of the yeast-derived CCT6 subunit homo-oligomeric ring complex prepared using an affinity grid and the average result of two-dimensional classification. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] It should be understood that the following examples are only exemplary illustrations and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect. Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available products, or can be prepared by known methods. If the specific technology or conditions are not specified in the examples, they are all conventional methods or carried out according to the technology or conditions described in the literature of the field, or according to the product instructions. The reagents and instruments used, etc., which do not indicate the manufacturer, are all conventional products that can be purchased through regular channels.

[0037] According to a preferred embodiment of the present invention, an electron microscope grid covered with a graphene film, a graphene oxide film or a carbon film is immersed in a pyr-NHS solution for reaction; the grid is then cleaned and dried to obtain a grid with pyr-NHS attached to the surface; the pyr-NHS on the surface of the obtained grid is used as a crosslinker, and the NZ-1 antibody solution is dripped onto the surface of the grid and incubated to achieve the fixation of the NZ-1 antibody protein on the surface of the grid. After the affinity grid is prepared, it can be directly used to specifically enrich the target protein from the cell lysate, including the following steps:

[0038] (1) Place the metal support with the grid in the cell culture plate, fix the affinity grid in the slot of the metal support, add the crude cell lysis solution containing the target protein to the well of the cell culture plate until the affinity grid is completely covered; (2) Incubate at 4-25°C for 20-60 minutes, and use a pipette to gently blow and mix the crude cell lysis solution every 5-10 minutes to allow the affinity grid to fully bind to the target protein; (3) After the incubation is completed, remove the affinity grid from the metal support and use a buffer solution to clean the surface of the grid to remove non-specifically bound proteins. This cleaning process also uses a hydrophobic matrix (such as a sealing film or glass slide) to carry the buffer solution. 30-50 μL of protein buffer is added dropwise to the hydrophobic matrix. The side of the grid anchored with the target protein is briefly in contact with the surface of the droplet, and then the droplet is absorbed along the side edge of the grid with filter paper. This cleaning step is repeated 2-3 times. At this point, the affinity grid anchored with the target protein is prepared and the typical cryo-EM sample preparation process can be immediately carried out. Taking the Vitrobot Mark IV rapid freezing sample preparation instrument as an example, 2 to 3 μL of buffer is dripped onto the surface of the grid, and the Vitrobot Mark IV is used to absorb the excess liquid on the grid to form an ultra-thin liquid film, and then the grid is quickly frozen by liquid ethane cooled by liquid nitrogen. The frozen grid can be used for cryo-EM data collection and high-resolution structure analysis.

[0039] Example 1 Preparation of affinity carrier network and characterization of surface properties

[0040] This embodiment provides a method for preparing an affinity grid for cryo-electron microscopy, and the preparation process is as follows: Figure 1 As shown, the specific preparation process is as follows:

[0041] 1) Small molecule crosslinker pyr-NHS modified graphene oxide network

[0042] Dissolve 4 mg of pyr-NHS in 10 mL of organic solvent methanol at room temperature overnight. Take an electron microscope grid with a carrier film such as graphene oxide and soak it in a methanol solution of pyr-NHS for 1 hour at room temperature 25°C. Then use the organic solvent methanol to clean the surface of the grid three times. The specific steps are as follows: add 10 μL of methanol to the side of the grid covered with the carrier film each time, and use a filter paper to absorb the surface liquid from the side edge of the grid to remove excess pyr-NHS. The cleaned grid is naturally air-dried at room temperature for 15 minutes.

[0043] The surface properties of the pyr-NHS-modified graphene oxide network prepared in this example are as follows: Figure 2 As shown in A. Among them, Figure 2A is the characteristic peak of the N1s element in the X-ray photoelectron spectrum of the graphene oxide grid modified with the small molecule crosslinker pyr-NHS (right) and the unmodified graphene oxide grid (left). A new characteristic peak of the N1s element on the surface of the graphene oxide grid modified with pyr-NHS appears, indicating that the small molecule pyr-NHS is successfully bound to the surface of the graphene oxide grid, and the binding of pyr-NHS changes the surface properties of the graphene oxide grid.

[0044] 2) Antibody protein NZ-1Fab is fixed on the surface of graphene oxide grid via pyr-NHS

[0045] The graphene oxide grid modified with pyr-NHS can be used to fix antibody protein. The antibody protein used in this embodiment is the Fab segment of NZ-1 antibody, which can specifically bind to the target protein expressed with PA tag fusion. The specific steps are as follows: (1) Dilute NZ-1Fab to a concentration of 80μg / mL using PBS buffer; (2) Take 5μL of NZ-1Fab solution and add it dropwise to the surface of the graphene oxide grid modified with pyr-NHS, and incubate at room temperature for 45min; (3) Use protein buffer (50mMTris-HCl pH 7.5, 100mM NaCl, 0.5mM EDTA, 1mM DTT) to clean the grid surface to terminate the cross-linking reaction of pyr-NHS to NZ-1Fab and remove excess NZ-1Fab. In this cleaning process, a hydrophobic sealing membrane is used to carry the protein buffer. 30 μL of the protein buffer is dripped onto the sealing membrane. The side of the grid fixed with NZ-1Fab is briefly brought into contact with the surface of the droplet, and then the droplet is absorbed along the side edge of the grid with filter paper. This cleaning step is repeated 3 times to prepare the affinity grid.

[0046] The effect of the modified graphene oxide grid on the fixation of the antibody NZ-1Fab in this example is as follows: Figure 2 B and C. Among them, Figure 2 B is a 120kV transmission electron microscope photo of negatively stained samples of graphene oxide grid (left) and affinity grid (right). Figure 2 C is the atomic force microscope (AFM) three-dimensional height morphology of the graphene oxide grid (left) and the affinity grid (right). The results show that the antibody NZ-1Fab was successfully immobilized on the surface of the pyr-NHS-modified graphene oxide grid. The concentration range of the antibody protein was 25-100μg / ml. Figure 2 D is a 120kV transmission electron microscopy photograph of negatively stained samples of 25μg / ml, 50μg / ml, and 100μg / ml NZ-1Fab fixed on the affinity grid.

[0047] Example 2 The affinity network is used to directly bind the target protein in cell lysate

[0048] The present invention displays TBCA (Tubulin-binding cofactor A) with a PA tag at the N-terminus on apoferritin (protein sequence diagram as shown in Figure 3 A), forming a TBCA-apoferritin complex. Taking this as an example, the specific process of using affinity carrier to directly affinity target protein from cell lysate and freeze sample preparation is as follows.

[0049] 1) Cell lysate processing flow

[0050] The expression plasmid of TBCA-apoferritin with PA tag was transformed into E. coli system for induction of expression. 6L of E. coli cultured in LB medium was cultured at 37°C until the OD600 value was 1.2, and 0.2mM IPTG was added to induce expression at 16°C for 18h and then centrifuged to collect cells. The collected cell pellet was crushed using a ball mill and fully dissolved with 100mL lysis buffer (50mMTris-HCl pH 7.5, 100mM NaCl, 0.5mM EDTA, 1mM DTT, protease inhibitor) at 4°C. The cell lysate was then centrifuged at 18,000rpm for 30min, and the supernatant was ultracentrifuged at 150000g for 2h to completely remove cell debris. Take the supernatant of the cell lysate after centrifugation, use a 15mL 100kDa concentrator to concentrate it to a volume of 3mL, and then perform glycerol density gradient centrifugation (glycerol concentration is 10%-40%, centrifugal speed is 41,000rpm, and time is 15h) to roughly separate proteins with different sedimentation coefficients. After centrifugation, sample the samples from low to high according to the glycerol gradient, and perform SDS-PAGE electrophoresis on the samples to determine the approximate tube number where the target protein is located. Figure 3 As shown in B, the TBCA-apoferritin target protein is present in the fraction with a glycerol concentration of 30%-40%; meanwhile, negatively stained samples were examined by electron microscopy ( Figure 3 C) shows that there are still a lot of impurities in the crude treatment solution.

[0051] 2) Affinity grid direct affinity target protein from crude cell lysis solution and frozen sample preparation process

[0052] like Figure 4As shown in the figure, the affinity grid is used to specifically bind the TBCA-apoferritin target protein in the crude cell lysis solution and the freezing sample preparation process is as follows: the grid metal support is placed in the well of the 6-well cell culture plate, and the affinity grid is fixed in the card slot of the metal support; the crude cell lysis solution containing TBCA-apoferritin (volume is 5mL) is added to the well of the cell culture plate to completely cover the affinity grid; incubate at room temperature (~25℃) for 20min, and use a pipette to gently blow the liquid every 5min to allow the affinity grid to fully bind to the TBCA-apoferritin with the PA tag. After the incubation is completed, the affinity grid is removed from the metal support using special tweezers for freezing sample preparation, and the surface of the grid is washed with protein buffer (50mMTris-HCl pH7.5, 100mM NaCl, 0.5mM EDTA, 1mM DTT) to remove non-specific binding proteins. This cleaning process also uses a hydrophobic sealing film to carry buffer. 30 μL of protein buffer is dripped onto the sealing film. The side of the grid anchored with TBCA-apoferritin is briefly in contact with the droplet surface, and then the droplet is absorbed along the side edge of the grid with filter paper. This cleaning step is repeated 3 times. The affinity grid anchored with TBCA-apoferritin protein is then directly used for typical cryo-EM sample preparation: Taking Vitrobot Mark IV as an example, 2.5 μL of protein buffer is dripped onto the surface of the affinity grid fixed with the target protein, and the excess liquid is sucked off using Vitrobot, and then the grid is quickly frozen by liquid ethane cooled with liquid nitrogen for subsequent cryo-EM data collection and three-dimensional structure analysis.

[0053] Cryo-electron microscopy was used to compare affinity grids and untreated graphene oxide grids / continuous carbon film grids ( Figure 5 ), it was found that the untreated graphene oxide grid ( Figure 5 A, left) and continuous carbon film grid ( Figure 5 B, left) After incubation directly with TBCA-apoferritin cell lysate, the sample background is messy and cannot be used for cryo-EM structure analysis; while using graphene oxide support film grid ( Figure 5 A, right) and continuous carbon film grids ( Figure 5 B, right) The prepared affinity network has obvious specific affinity for the target protein TBCA-apoferritin, with a clean background, and can be used for subsequent cryo-electron microscopy data collection and high-resolution structure analysis.

[0054] Example 3: Application of the above affinity grid-prepared frozen sample to cryo-EM high-resolution structural studies

[0055] The frozen samples, which were specifically enriched from the crude cell culture fluid using affinity grids, were then used for high-resolution structural elucidation studies using cryo-EM. Data were collected on a Titan Krios cryo-EM equipped with a K3 camera at an accelerating voltage of 300 kV. The pixel size of the data collection was The defocus value range is -0.8 to -2.5m, and the total electron dose per photo is about The overall resolution was obtained by single particle reconstruction analysis. The three-dimensional reconstructed density map of Figure 6 A is the three-dimensional reconstructed electron density map of TBCA-apoferritin. Figure 6 B is a high-resolution density map of the representative amino acid side chains of apoferritin in TBCA-apoferritin. Figure 6 C. By adjusting the density threshold, the density of TBCA protein can be clearly seen in the reconstruction result of the frozen sample prepared by affinity grid (right). However, in the three-dimensional reconstruction result of the TBCA-apoferritin frozen sample prepared by conventional freezing sample preparation process using ordinary graphene oxide grid (left), the density of the TBCA part is seriously missing, indicating that the gas-liquid interface tension caused serious damage to the complex during the freezing sample preparation process. The use of affinity grid to anchor the target protein to the grid surface protects the protein complex to a certain extent, making the structure of the complex more complete.

[0056] Example 4: The frozen sample prepared by the affinity grid solves the problem of protein dominant orientation

[0057] 1) Expression and purification of homo-oligomeric complex protein of CCT6 subunit of yeast TRiC complex

[0058] The present invention inserts a PA tag between the 285th and 286th amino acids of the CCT6 expression sequence, adds a 6xHis tag and a TEV restriction site at the N-terminus, constructs a pET28a expression vector and transforms it into an E. coli system to induce expression, and culture 6L of E. coli cultured in LB medium at 37°C until the OD600 value is 1.2, adds 0.2mM IPTG at 16°C to induce expression for 18h, and then collects the cells by centrifugation. The collected cell precipitate is crushed using a ball mill, and fully dissolved with 100ml lysis buffer (20mMHEPES / KOH pH 7.4, 300mM NaCl, 10mM MgCl2, 10% glycerol, 5mMβ-ME, 2mM ATP, protease inhibitor) at 4°C. The cell lysate is then centrifuged at 18,000rpm for 30min, and the supernatant is ultracentrifuged at 200000g for 1.5h to completely remove cell debris. The supernatant was incubated with a nickel column at 4°C for 30 min, and the target protein was eluted with a buffer containing 300 mM imidazole. The eluate was concentrated by centrifugation using a 100 kDa concentrator and then subjected to glycerol density gradient centrifugation (glycerol concentration was 10%-40%, centrifugal speed was 41,000 rpm, and time was 15 h). After centrifugation, the samples were sampled from low to high according to the glycerol gradient, and the samples were subjected to SDS-PAGE electrophoresis to determine the tube number where the target protein was located, and the protein was concentrated using a 100 kDa concentrator.

[0059] 2) Using affinity grids to prepare frozen samples can solve the problem of dominant orientation of some protein samples.

[0060] The homologous oligomeric complex protein of the CCT6 subunit of the yeast TRiC complex was frozen using a common holey carbon grid, which showed a very serious problem of dominant orientation. From the original cryo-EM image of the sample and the results of two-dimensional classification, it can be seen that the distribution of the protein complex is basically all apical orientation, which seriously affects the study of high-resolution structural analysis of cryo-EM ( Figure 7 A). When the affinity grid was used to prepare frozen samples of protein complexes, a large number of protein particle images with lateral and tilted distributions were clearly observed from both the original images and the results of two-dimensional classification ( Figure 7 B), which indicates that the sample prepared by the affinity grid provided by the present invention can overcome the problem of dominant orientation and be used for high-resolution structural analysis.

[0061] The affinity grid preparation method provided in the embodiment of the present invention and the method for freezing the target protein in the affinity cell lysate to achieve direct affinity of the target protein from the cell lysate for freezing and high-resolution cryo-electron microscopy structure analysis, simplifying the protein purification process. The present invention non-covalently modifies the surface of the grid by the organic small molecule pyr-NHS to achieve the fixation of the antibody protein, which is simple to operate and can be applied to the reaction system based on affinity such as antigen-antibody to achieve the anchoring of different proteins (as shown in Example 1). Furthermore, the freezing sample preparation method of an affinity grid provided in the present invention for cryo-electron microscopy research realizes the specific enrichment of the target protein directly from the cell lysate by the affinity grid (as shown in Example 2), greatly simplifies the purification process of the protein complex before the preparation of the frozen sample, which has an enlightening effect on the freezing sample preparation of large complexes that are difficult to purify. The affinity grid freezing sample preparation method provided in the embodiment of the present invention can be applied to the work of high-resolution structure analysis of cryo-electron microscopy, and the frozen sample prepared using the affinity grid can be reconstructed to obtain the high-resolution structure of the complex through the conventional single particle analysis process. At the same time, the embodiment of the present invention uses an affinity grid to prepare frozen samples, and the target protein can be fixed on the surface of the grid through antibody proteins, etc., which solves the problem of protein denaturation caused by the surface tension of the gas-liquid interface damaging the protein complex to a certain extent. The strategy of the affinity grid makes the structure of the protein complex more complete (as shown in Example 3). The optimization process of the frozen sample preparation conditions during the frozen sample preparation process is saved. At the same time, the use of the affinity grid provided in the present invention for frozen sample preparation can solve the problem of the dominant orientation of the protein complex in the frozen sample, so that the frozen sample can be directly used for subsequent cryo-electron microscopy high-resolution structural analysis research. Compared with the traditional method of using detergents in the frozen sample preparation process, the process of exploring and optimizing the sample preparation conditions is greatly simplified; at the same time, compared with the strategy of using a tilting sample stage during data collection, the process of data collection and subsequent data processing is simplified (as shown in Example 4).

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An affinity grid for cryo-electron microscopy, characterized in that: It includes a carbonaceous carrier film covering a carrier mesh, an organic small molecule pyr-NHS connected to the carbonaceous carrier film, and a specific antibody protein connected to the organic small molecule pyr-NHS; the carbonaceous carrier film is a graphene film, an oxidized graphene film or a carbon film.

2. The affinity grid for cryo-electron microscopy according to claim 1, characterized in that The specific antibody protein is NZ-1 antibody protein.

3. The affinity grid for cryo-electron microscopy according to claim 1 or 2, characterized in that The affinity carrier network is prepared by a method comprising the following steps: using an organic small molecule pyr-NHS to fix the NZ-1 antibody protein on the surface of the carbonaceous carrier film covering the carrier network.

4. A method for preparing an affinity grid for cryo-electron microscopy, characterized in that: The following steps are involved: The affinity grid is prepared by fixing the specific antibody protein on the surface of the carbonaceous carrier film covering the grid using the organic small molecule pyr-NHS; the carbonaceous carrier film is a graphene film, a graphene oxide film or a carbon film; the specific antibody protein is preferably the NZ-1 antibody protein.

5. The method for preparing an affinity grid for cryo-electron microscopy according to claim 4, characterized in that: include: 1) immersing a grid covered with a graphene film, a graphene oxide film or a carbon film in a pyr-NHS solution for reaction; 2) cleaning and drying the carrier mesh treated in step 1) to obtain a carrier mesh with pyr-NHS attached to the surface; 3) Using the pyr-NHS on the surface of the grid obtained in step 2) as a cross-linking agent, the NZ-1 antibody solution is coated on the surface of the grid and incubated to obtain an affinity grid.

6. Use of the affinity grid for cryo-electron microscopy described in any one of claims 1-3 or the affinity grid obtained by the preparation method of the affinity grid for cryo-electron microscopy described in claim 4 or 5 in preparing frozen samples for cryo-electron microscopy; preferably, the affinity grid is used to directly and specifically enrich the target protein containing the PA tag from the cell lysate.

7. A method for preparing a frozen sample, characterized in that: include: The organic small molecule pyr-NHS is used to fix the NZ-1 antibody protein on the surface of the carbonaceous carrier film covering the carrier network to prepare an affinity carrier network; the affinity carrier network is incubated with a cell lysate containing the target protein, so that the affinity carrier network has affinity to the target protein from the cell lysate, and is directly frozen to prepare a sample; the target protein in the cell lysate is a target protein fused with an expression PA tag, and the carbonaceous carrier film is a graphene film, an oxidized graphene film or a carbon film.

8. The method for preparing a frozen sample according to claim 7, characterized in that: The method comprises subjecting a cell lysate to high-speed centrifugation and glycerol density gradient centrifugation to obtain a crude cell lysate containing a target protein fused with an expression PA tag; fixing the affinity grid in a cell culture plate, adding the crude cell lysate to the cell culture plate for incubation, and then freezing the plate for sample preparation.

9. Application of the frozen sample obtained by the method for preparing a frozen sample according to any one of claims 7 to 8 in the structural analysis of biological macromolecules, wherein the application is any one of the following: 1) Avoid damage to biomacromolecules caused by the gas-liquid interface during sample preparation; 2) Overcoming the orientation tendency problem of biological sample distribution; 3) Achieve affinity of target protein directly from cell lysate for frozen sample preparation; 4) Collect cryo-EM data and conduct high-resolution cryo-EM structure analysis.

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