Affinity grid for cryo-electron microscopy and method for preparing frozen sample thereof and use thereof

Abstract

Provided in the present invention are an affinity grid for cryo-electron microscopy and a method for preparing a frozen sample thereof and the use thereof. The affinity grid comprises a carbonaceous bearing film covering the grid, organic small molecule pyr-NHS linking to the carbonaceous bearing film, and a specific antibody protein linking to pyr-NHS, wherein the carbonaceous bearing film is a graphene film, a graphene oxide film or a carbon film. The affinity grid having the carbonaceous bearing film modified with organic small molecule pyr-NHS of the present invention has the effect of efficiently enriching low-abundance target proteins. Preferably, the specific antibody protein is anchored to the surface of the grid by using the organic small molecule pyr-NHS, and the obtained affinity grid can realize efficient affinity purification and fusion expression of a target protein having a specific tag directly from a cell lysis buffer so as to directly prepare a frozen sample and perform structural analysis of high-resolution cryo-electron microscopy. The present invention reduces the time and steps of sample purification, improves sample integrity, reduces a demanded sample size, and solves the preferred orientation problem of sample distribution.

Description

Affinity grid for cryo-electron microscopy and preparation method and application of frozen samples thereof

[0001] Cross-references

[0002] This application claims priority to Chinese patent application No. 202311134981.4, filed on September 4, 2023, entitled “Affinity grid for cryo-electron microscopy and preparation method and application of frozen samples thereof,” all disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0003] 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

[0004] Since its emergence in the 1970s, cryo-electron microscopy (cryo-EM) has undergone a long period of development, particularly driven by technological innovations in hardware and software over the past decade or so. It has become the most important means of analyzing the near-atomic resolution structures of biomacromolecules, significantly advancing research efforts to elucidate biological mechanisms at the molecular level. First, a biological sample is dripped onto a support film-covered grid and then rapidly frozen in liquid ethane or propane cooled by liquid nitrogen to form a glassy, ​​amorphous ice layer. This maintains the biomacromolecules in a near-physiological state within the frozen sample. The presence of a certain thickness of glassy ice also reduces radiation damage to the biomacromolecules from the electron beam. Finally, data collection and computational reconstruction of the frozen sample using a transmission electron microscope (TEM) are used to obtain the three-dimensional structure of the biomacromolecules.

[0005] With the continuous advancement of technology in recent years, electron microscopy, detector technology, and image data processing algorithms are no longer the obstacles that hinder cryo-EM structural analysis. However, cryo-EM still faces some bottlenecks that severely limit the efficiency of using this technology to analyze biomolecular structures. One of the key 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. Currently, the main problems with frozen sample preparation include: the difficulty in obtaining samples of biological macromolecules and super-macromolecular complexes, damage to biological samples caused by the gas-liquid interface during sample preparation, the orientation bias of biological sample distribution, and the extremely tedious biological sample purification process in the early stages of cryo-EM sample preparation. These problems have led to many structural studies requiring a large amount of time and effort to be invested in sample optimization.

[0006] Summary of the Invention

[0007] 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 orientation tendency of sample distribution 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 affinity cell lysate with a target protein with a tag such as PA or FLAG to prepare a frozen sample.

[0008] In a first aspect, the affinity grid for cryo-electron microscopy provided by the present invention includes a carbonaceous carrier film covering the grid, 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, a graphene oxide film or a carbon film.

[0009] In the present invention, the hybrid state of the organic small molecule pyr-NHS with a conjugated structure and the carbon atoms covered on the surface of the support grid through π-π stacking interaction is sp 2 Hybrid graphene, graphene oxide or carbon film are closely combined to the above sp 2The surface of the hybridized specific carbonaceous carrier membrane is modified with non-covalent bonds. 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 terminus, anchoring the specific antibody protein to the carrier surface. The affinity carrier obtained by the present invention can directly affinity-fuse the target protein expressing a specific tag from the cell lysate for frozen sample preparation and high-resolution cryo-electron microscopy structure analysis, simplifying 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 frozen sample preparation for cryo-electron microscopy research, and can achieve the specific enrichment of the target protein directly from the cell lysate by the affinity carrier, greatly simplifying 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 membrane with oxygen plasma, nor is there any need to use a buffer solution with special components to treat the carrier mesh. The specific affinity grid provided by the present invention can better anchor the target protein to the grid surface, particularly overcoming the problems of cumbersome protein purification processes, protein fragility caused by the gas-liquid interface, and distribution orientation bias. While improving the success rate of frozen sample preparation, the present invention also enhances the quality of frozen samples prepared using the affinity grid and improves sample preparation efficiency, enabling the reconstruction of high-resolution structures of complexes using conventional single-particle analysis procedures. Frozen samples prepared directly from cell lysates using the affinity grid exhibit a clean background, making them more suitable for subsequent cryo-electron microscopy data collection and high-resolution structural analysis.

[0010] Preferably, the specific antibody proteins include, but are not limited to, NZ-1 antibody protein, anti-FLAG tag M2 antibody protein, HA tag antibody, HIS tag antibody, and anti-GFP antibody. In the present invention, not only the PA-NZ-1 affinity system and the FLAG-M2 affinity system are effective, but other high-affinity protein interaction systems are also effective. This invention uses NZ-1 antibody protein and M2 antibody protein as examples for verification.

[0011] Further preferably, the affinity grid is prepared by a method comprising the following steps: covering the surface of the carbonaceous support film on the grid with the organic small molecule pyr-NHS to prepare the affinity grid; preferably, the specific antibody protein is anchored on the surface of the carbonaceous support film covering the grid using the organic small molecule pyr-NHS.

[0012] In a second aspect, the present invention provides an affinity grid for cryo-electron microscopy, comprising a carbonaceous support membrane covering the grid and a small organic molecule, pyr-NHS, bonded to the carbonaceous support membrane; the carbonaceous support membrane can be a graphene membrane, a graphene oxide membrane, or a carbon membrane. Research by the present invention has found that bonding the small organic molecule, pyr-NHS, to the carbonaceous support membrane also has the effect of enriching proteins.

[0013] In a third aspect, the present invention provides a method for preparing an affinity grid for cryo-electron microscopy, comprising the following steps: covering the surface of a carbonaceous carrier film on the grid with an organic small molecule pyr-NHS to obtain an affinity grid; preferably, using the organic small molecule pyr-NHS to anchor specific antibody proteins on the surface of the 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.

[0014] Preferably, the method for preparing the affinity grid for cryo-electron microscopy includes:

[0015] 1) Immersing a grid covered with a graphene film, a graphene oxide film or a carbon film in a pyr-NHS solution for reaction.

[0016] 2) Cleaning and drying the grid treated in step 1) to obtain a grid with pyr-NHS attached to the surface.

[0017] 3) Using the pyr-NHS on the surface of the grid obtained in step 2) as a cross-linking agent, the specific antibody solution is coated on the surface of the grid and incubated to obtain an affinity grid.

[0018] Further preferably, in step 1), the concentration of the pyr-NHS solution is 0.8 to 1.2 mM; the immersion temperature is 10 to 30° C., and the immersion time is 45 min to 2 h; preferably, the step comprises dissolving pyr-NHS in a first organic solvent to obtain the pyr-NHS solution; the first organic solvent is preferably DMF or methanol.

[0019] Further preferably, in step 2), the cleaning step comprises: dripping a second organic solvent onto the carrier mesh, then absorbing the surface liquid from the side edge of the carrier mesh, and repeating the cleaning step 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.

[0020] Further preferably, step 3) comprises dropping 5 to 10 μL of a specific 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 in which the specific antibody is anchored to the surface of the grid; preferably, the solvent of the specific antibody solution is a buffer solution that does not contain an amino group, preferably a PBS buffer solution, an acetate buffer solution or a HEPES buffer solution; after the incubation, the step of washing the surface of the grid with a second buffer solution is preferably further included; the second buffer solution is a buffer solution containing an amino group, preferably a Tris buffer solution or a glycine solution.

[0021] 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 a specific tag from the cell lysate.

[0022] In a fourth aspect, the present invention provides a method for preparing frozen samples, comprising: covering the surface of a carbonaceous carrier film on a carrier grid with an organic small molecule pyr-NHS, preferably using the organic small molecule pyr-NHS to anchor a specific antibody protein on the surface of the carbonaceous carrier film covering the carrier grid to prepare an affinity carrier grid; incubating the affinity carrier grid with a cell lysate containing a target protein, so that the affinity carrier grid is affinity-bound to the target protein from the cell lysate, and directly freezing the sample; the target protein in the cell lysate adopts a target protein fused with a specific expression tag, and the carbonaceous carrier membrane is a graphene film, a graphene oxide film or a carbon film.

[0023] Preferably, the method for preparing the frozen sample comprises subjecting the 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 specific expression tag; fixing the affinity grid in a cell culture plate, and adding the crude cell lysis solution to the cell culture plate for incubation.

[0024] Further preferably, the method for preparing the frozen sample comprises centrifuging the cell lysate at 10,000-50,000 g for 15-45 min, then ultracentrifuging at 100,000-200,000 g for 1-3 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 4-25° C. for 20-60 min, preferably, using a pipette to mix the crude cell lysis solution every 5-10 min to allow the affinity grid to bind to the target protein.

[0025] 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 the structure of biological macromolecules, wherein the application is any of the following.

[0026] 1) Avoid damage to biomacromolecules caused by the gas-liquid interface during sample preparation.

[0027] 2) Enrichment of low-abundance target proteins.

[0028] 3) Overcoming the orientation tendency problem of biological sample distribution.

[0029] 4) Achieve affinity of target protein directly from cell lysate for frozen sample preparation.

[0030] 5) Conduct cryo-EM data collection and high-resolution cryo-EM structure analysis.

[0031] According to a preferred embodiment of the present invention, the high-affinity PA-NZ-1 affinity system was used as an example to demonstrate the excellent application performance of the affinity network of the present invention. However, applicable affinity systems include, but are not limited to, the PA-NZ-1 affinity system and can also be applied to other high-affinity protein interaction systems, such as the FLAG-M2 affinity system and the HA-HA tag antibody affinity system.

[0032] 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 through π-π stacking interaction with the carbon atoms covered on the surface of the carrier network. 2 The hybridized graphene, graphene oxide or carbon film is tightly combined, and its surface is modified by non-covalent bonds. 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, anchoring the specific antibody to the surface of the grid, thereby obtaining an affinity grid. The affinity grid fixed with the specific antibody can then be directly used to obtain the target protein with a specific 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 the 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 tedious 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 grid surface through antibodies, which to a certain extent overcomes the problems of protein fragility and distribution orientation tendency caused by the gas-liquid interface, and improves the success rate of frozen sample preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to 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 any creative work.

[0034] FIG1 is a schematic diagram of the preparation of the affinity grid provided in Example 1 of the present invention and its application in cell lysate specific affinity target protein and freezing sample preparation.

[0035] Figure 2 is a characterization 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 photograph 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 image of the graphene oxide grid (left) and the affinity grid (right); (D) is a 120kV transmission electron microscope photograph of the negatively stained sample of the graphene oxide grid covered with pyr-NHS treated with different concentrations of NZ-1 Fab.

[0036] Figure 3 is a 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 an SDS-PAGE electrophoresis Coomassie Brilliant Blue staining image of the crude cell lysis solution of TBCA-apoferritin with a PA label; (C) is a 120kV transmission electron microscope photograph of a negatively stained sample of the crude cell lysis solution of TBCA-apoferritin with a PA label.

[0037] FIG4 is a schematic diagram of the process of preparing the affinity grid provided in Example 2 of the present invention and applying it to directly bind the target protein in cell lysate and perform freezing sample preparation.

[0038] Figure 5 is a 200kV 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 200kV cryo-electron micrograph of the graphene oxide supported membrane grid (left) and the graphene oxide supported membrane affinity grid (right) incubated with cell lysate to prepare samples; (B) is a 200kV cryo-electron micrograph of the continuous carbon film grid (left) and the continuous carbon film affinity grid (right) incubated with cell lysate to prepare samples.

[0039] Figure 6 shows the affinity grid frozen sample provided in Example 3 of the present invention 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 some 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 grid (left) and affinity grid frozen sample preparation (right).

[0040] Figure 7 shows the application of the affinity grid provided in Example 4 of the present invention to overcome the problem of preferential orientation of protein samples; Figure 7(A) is a cryo-electron microscopy photograph and two-dimensional classification average result of a yeast-derived CCT6 subunit homo-oligomeric ring complex prepared using an ordinary holey carbon R2 / 1 copper grid; Figure 7(B) is a cryo-electron microscopy photograph and two-dimensional classification average result of a yeast-derived CCT6 subunit homo-oligomeric ring complex prepared using an affinity grid.

[0041] Figure 8 shows the application of pyr-NHS-modified graphene oxide grid for efficient enrichment of low-abundance target proteins provided in Example 5 of the present invention; Figure 8(A) is a cryo-electron micrograph of a yeast-derived CCT6 homo-oligomeric ring complex prepared using an ordinary unsupported membrane holey carbon grid; Figure 8(B) is a cryo-electron micrograph of a CCT6 homo-oligomeric ring complex prepared using an unmodified graphene oxide grid; Figure 8(C) is a cryo-electron micrograph of a CCT6 homo-oligomeric ring complex prepared using a pyr-NHS-modified graphene oxide grid.

[0042] Figure 9 shows a cryo-electron micrograph of the frozen sample prepared by anchoring the M2 antibody on the surface of the graphene oxide grid via pyr-NHS and affinity with the target protein CCT6-FLAG TRiC complex as provided in Example 6 of the present invention, and a comparative experiment thereof; Figure 9 (A) is a cryo-electron micrograph of CCT6-FLAG TRiC prepared using an unmodified graphene oxide grid; Figure 9 (B) is a cryo-electron micrograph of CCT6-FLAG TRiC prepared using an affinity grid of the M2 antibody anchored on a pyr-NHS-modified graphene oxide grid. DETAILED DESCRIPTION

[0043] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0044] It should be understood that the following examples are merely illustrative 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 encompassed within the scope that the present invention is intended to protect. Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. Those in which specific techniques or conditions are not specified in the examples are all conventional methods or are carried out according to the techniques or conditions described in the literature in this field, or are carried out according to the product instructions. Those whose manufacturers are not specified for the reagents and instruments used are all conventional products that can be purchased through regular channels.

[0045] 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 obtained grid surface is used as a cross-linking agent, and a specific antibody (protein) solution is added dropwise to the grid surface and incubated to achieve anchoring of the specific antibody protein to the grid surface. 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: (1) Place the metal support on which the grid is placed in the cell culture plate, and 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 wash the surface of the grid to remove non-specifically bound proteins. This washing process also uses a hydrophobic matrix (such as sealing film or glass slide, etc.) 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 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 washing step is repeated 2-3 times. At this point, the affinity grid anchored with the target protein is complete, and the typical cryo-EM sample preparation process can be immediately carried out. Using the Vitrobot Mark IV rapid freezing instrument as an example, 2-3 μL of buffer is dripped onto the grid surface. The Vitrobot Mark IV removes excess liquid from the grid, forming an ultra-thin liquid film. The grid is then rapidly frozen using liquid ethane cooled by liquid nitrogen. The frozen grid is then ready for cryo-EM data collection and high-resolution structural analysis.

[0046] According to a preferred embodiment of the present invention, the high-affinity PA-NZ-1 affinity system was used for assembly, demonstrating the excellent performance of the affinity network. However, applicable affinity systems include, but are not limited to, the PA-NZ-1 affinity system and can also be applied to other high-affinity interaction systems, such as the FLAG-M2 affinity system and the HA-HA tag antibody affinity system.

[0047] Example 1 Preparation and Surface Characterization of Affinity Grid

[0048] This embodiment provides a method for preparing an affinity grid for cryo-electron microscopy. The preparation process is shown in Figure 1, and the specific preparation process is as follows.

[0049] 1) Graphene oxide network modified with small molecule crosslinker pyr-NHS.

[0050] Dissolve 4 mg of pyr-NHS in 10 mL of methanol at room temperature overnight. Soak an electron microscope grid with a support film (e.g., graphene oxide) in the methanol solution for 1 hour at 25°C. Wash the grid three times with methanol: Add 10 μL of methanol to the side of the grid covered with the support film. Remove any excess pyr-NHS by blotting the surface with filter paper. Allow the cleaned grid to air-dry at room temperature for 15 minutes.

[0051] The surface properties of the pyr-NHS-modified graphene oxide grid prepared in this example are shown in Figure 2A. Figure 2A shows 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 figure) and the unmodified graphene oxide grid (left figure). A new characteristic peak of the N1s element appeared on the surface of the graphene oxide grid modified with pyr-NHS, indicating that the small molecule pyr-NHS was successfully bound to the surface of the graphene oxide grid, and the binding of pyr-NHS changed the surface properties of the graphene oxide grid.

[0052] 2) Antibody protein NZ-1 Fab was anchored to the surface of graphene oxide grid via pyr-NHS.

[0053] The pyr-NHS-modified graphene oxide grid can be used to anchor antibody proteins. The antibody protein used in this example is the Fab segment of the NZ-1 antibody, which can specifically bind to the target protein expressed with a PA tag. The specific steps are as follows: (1) Dilute the NZ-1 Fab to a concentration of 80 μg / mL using PBS buffer; (2) Take 5 μL of the NZ-1 Fab solution and add it dropwise to the surface of the pyr-NHS-modified graphene oxide grid and incubate it at room temperature for 45 minutes; (3) Use protein buffer (50mM Tris-HCl pH 7.5, 100mM NaCl, 0.5mM EDTA, 1mM DTT) to wash the grid surface to terminate the cross-linking reaction of pyr-NHS to NZ-1 Fab and remove excess NZ-1 Fab. In this cleaning process, a hydrophobic sealing film is used to carry the protein buffer. 30 μL of protein buffer is added dropwise to the sealing film. The side of the grid fixed with NZ-1 Fab is briefly brought into contact with the surface of the droplet. After that, the droplet is absorbed along the side edge of the grid with filter paper. This cleaning step is repeated three times to prepare the affinity grid.

[0054] The anchoring effect of the modified graphene oxide grid on the antibody NZ-1 Fab in this embodiment is shown in Figures 2B and C. Among them, Figure 2B is a 120kV transmission electron microscopy photo of the negatively stained samples of the graphene oxide grid (left picture) and the affinity grid (right picture), and Figure 2C is an atomic force microscope (AFM) three-dimensional height morphology image of the graphene oxide grid (left picture) and the affinity grid (right picture). The results all show that the antibody NZ-1 Fab is successfully anchored to the surface of the pyr-NHS-modified graphene oxide grid. The concentration range of the antibody protein is 25-100μg / ml, and Figure 2D is a 120kV transmission electron microscopy photo of the negatively stained samples of 25μg / ml, 50μg / ml, and 100μg / ml NZ-1 Fab anchored to the affinity grid.

[0055] Example 2: The affinity network is used to directly bind the target protein to the cell lysate

[0056] In this study, TBCA (Tubulin-binding cofactor A) with a PA tag at its N-terminus is displayed on apoferritin (see Figure 3A for a schematic diagram of the protein sequence), forming a TBCA-apoferritin complex. Using this as an example, the affinity network was used to directly bind the target protein from cell lysate and then freeze-sample preparation as follows.

[0057] 1) Cell lysate processing procedure.

[0058] The PA-tagged TBCA-apoferritin expression plasmid was transformed into E. coli to induce expression. Six liters of E. coli cultured in LB medium were grown at 37°C to an OD600 of 1.2. 0.2 mM IPTG was added for induction at 16°C for 18 hours, and the cells were harvested by centrifugation. The collected cell pellet was disrupted using a ball mill and thoroughly lysed with 100 mL of lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, and protease inhibitors) at 4°C. The cell lysate was then centrifuged at 18,000 rpm for 30 minutes, and the supernatant was ultracentrifuged at 150,000 g for 2 hours to completely remove cell debris. After centrifugation, the supernatant of the cell lysate was concentrated to 3 mL using a 15 mL 100 kDa concentrator. Glycerol density gradient centrifugation (glycerol concentration of 10%-40%, centrifugation speed of 41,000 rpm, time of 15 hours) was then performed to crudely separate proteins with different sedimentation coefficients. After centrifugation, samples were divided into tubes according to the glycerol gradient from low to high, and SDS-PAGE electrophoresis was performed to determine the approximate tube number of the target protein. As shown in Figure 3B, the TBCA-apoferritin target protein was present in the fractions with glycerol concentrations of 30%-40%. Electron microscopy of negatively stained samples (Figure 3C) also revealed the presence of numerous contaminants in the crude solution.

[0059] 2) Affinity grid directly binds the target protein from the crude cell lysate and freezes the sample preparation process.

[0060] As shown in Figure 4, the process of using affinity grids to perform specific affinity binding of the TBCA-apoferritin target protein in the crude cell lysate and freeze-sample preparation is as follows: Place the grid metal holder in the well of a 6-well cell culture plate and fix the affinity grid in the slot of the metal holder; add crude cell lysate containing TBCA-apoferritin (volume 5 mL) to the well of the cell culture plate to completely cover the affinity grid; incubate at room temperature (~25°C) for 20 minutes, and gently pipette the liquid every 5 minutes to allow the affinity grid to fully bind to the PA-tagged TBCA-apoferritin. After incubation, use tweezers specially designed for freeze-sample preparation to remove the affinity grid from the metal holder and wash the grid surface with protein buffer (50mM Tris-HCl pH7.5, 100mM NaCl, 0.5mM EDTA, 1mM DTT) to remove non-specifically bound proteins. This cleaning process also uses a hydrophobic sealing film to hold the buffer. 30 μL of protein buffer is dripped onto the sealing film. The side of the grid anchored with TBCA-apoferritin is briefly brought into contact with the droplet surface, and the droplet is then blotted off along the side edge of the grid with filter paper. This cleaning step is repeated three times. The affinity grid anchored with TBCA-apoferritin protein is then directly used for typical cryo-EM sample preparation: using the Vitrobot Mark IV as an example, 2.5 μL of protein buffer is dripped onto the surface of the affinity grid immobilized with the target protein. The Vitrobot is used to aspirate the excess liquid, and the grid is then rapidly frozen using liquid ethane cooled by liquid nitrogen for subsequent cryo-EM data collection and 3D structure elucidation.

[0061] Cryo-electron microscopy was used to compare the affinity grids and untreated graphene oxide grids / continuous carbon film grids (Figure 5). It was found that the background of the samples of the untreated graphene oxide grid (Figure 5A, left) and the continuous carbon film grid (Figure 5B, left) directly incubated with the crude TBCA-apoferritin cell lysate was messy and could not be used for cryo-electron microscopy structure analysis; while the affinity grids prepared using graphene oxide supported membrane grids (Figure 5A, right) and continuous carbon film grids (Figure 5B, right) had obvious specific affinity for the target protein TBCA-apoferritin, with a clean background, and could be used for subsequent cryo-electron microscopy data collection and high-resolution structure analysis.

[0062] Example 3: Application of the above-mentioned affinity grid-prepared frozen sample for cryo-electron microscopy high-resolution structural studies

[0063] The frozen samples, which were specifically enriched from the crude cell culture medium using affinity grids, were then used for high-resolution structural analysis 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 -2.5 μm, and the total electron dose per photo is approximately The overall resolution was obtained by single particle reconstruction analysis. Figure 6A shows the 3D reconstructed electron density map of TBCA-apoferritin, and Figure 6B shows the high-resolution density map of the representative amino acid side chains of apoferritin in TBCA-apoferritin. Furthermore, in Figure 6C, the density threshold is adjusted and the density of the TBCA protein is clearly visible in the reconstruction results of the frozen sample prepared using an affinity grid (right). However, the density of the TBCA portion is severely missing in the 3D reconstruction results of the frozen sample of TBCA-apoferritin prepared using a conventional freezing process using an ordinary graphene oxide grid (left). This indicates that the gas-liquid interfacial tension has severely damaged the complex during the freezing process. The use of an affinity grid to anchor the target protein to the grid surface protects the protein complex to a certain extent, making the complex structure more complete.

[0064] Example 4: Frozen samples prepared with the affinity grid solve the problem of protein orientation

[0065] 1) Expression and purification of the homo-oligomeric complex protein of the CCT6 subunit of the yeast TRiC complex.

[0066] The present invention inserts a PA tag between amino acids 285 and 286 of the CCT6 expression sequence, and simultaneously adds a 6xHis tag and a TEV cleavage site at the N-terminus. The pET28a expression vector is constructed and transformed into E. coli for induced expression. Six liters of E. coli cultured in LB medium are cultured at 37°C to an OD600 value of 1.2. 0.2 mM IPTG is then added to induce expression at 16°C for 18 hours, and the cells are then harvested by centrifugation. The collected cell pellet is disrupted using a ball mill and thoroughly lysed in 100 ml of lysis buffer (20 mM HEPES / KOH pH 7.4, 300 mM NaCl, 10 mM MgCl2, 10% glycerol, 5 mM β-ME, 2 mM ATP, and protease inhibitors) at 4°C. The cell lysate is then centrifuged at 18,000 rpm for 30 minutes, and the supernatant is then ultracentrifuged at 200,000 g for 1.5 hours 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 divided into tubes according to the glycerol gradient from low to high, 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.

[0067] 2) Using affinity grids to prepare frozen samples can solve the problem of dominant orientation of some protein samples.

[0068] Cryo-preparation of homo-oligomeric complex proteins of the CCT6 subunit of the yeast TRiC complex using a conventional Holy Carbon grid revealed a severe problem of preferential orientation. Raw cryo-EM images and two-dimensional classification results revealed that the protein complex was essentially oriented apically, severely hindering high-resolution cryo-EM structural analysis (Figure 7A). However, cryo-preparation of the protein complex using an affinity grid revealed a significant number of sideways and tilted distributions of protein particles, both in raw images and two-dimensional classification results (Figure 7B). This demonstrates that samples prepared using the affinity grid provided by the present invention can overcome this preferential orientation problem and facilitate high-resolution structural analysis.

[0069] The affinity grid preparation method provided in the embodiment of the present invention and the method for directly affinity freezing the target protein in the cell lysate achieves affinity directly from the cell lysate for freezing and performing high-resolution cryo-electron microscopy structure analysis, simplifying the protein purification process. The present invention uses the organic small molecule pyr-NHS to non-covalently modify the grid surface to achieve anchoring of antibody proteins. The operation is simple and can be applied to antigen-antibody and other affinity-based reaction systems to achieve anchoring of different proteins (as shown in Example 1). Furthermore, the affinity grid provided in the present invention is applied to the freezing method for cryo-electron microscopy research to achieve the specific enrichment of the target protein directly from the cell lysate by the affinity grid (as shown in Example 2), which greatly simplifies the purification process of the protein complex before the preparation of the frozen sample, which has a revelation effect on the freezing sample of large complexes that are difficult to purify. The affinity grid freezing method provided in the embodiment of the present invention can be applied to the work of high-resolution structure analysis of cryo-electron microscopy. 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 utilizes affinity grids to prepare frozen samples, and the target protein can be anchored to the grid surface through antibody proteins, etc., which solves the problem of protein denaturation caused by the damage of the surface tension of the gas-liquid interface to the protein complex to a certain extent. The strategy of affinity grids makes the structure of the protein complex more complete (as shown in Example 3). It saves the optimization process of frozen sample preparation conditions during frozen sample preparation. 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 structure 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 later data processing is simplified (as shown in Example 4).

[0070] Example 5 Organic small molecule pyr-NHS modified carbonaceous support membrane affinity grid can efficiently enrich low-abundance target protein

[0071] Figure 8 shows a cryo-electron microscopy photograph and a comparative experiment of a frozen sample preparation using a pyr-NHS-modified graphene oxide grid as an affinity grid provided by an embodiment of the present invention; the embodiment of the present invention explores the enrichment effect of a purified protein complex using a carbonaceous support membrane grid modified with an organic small molecule pyr-NHS as an affinity grid. For a low concentration of homo-oligomeric ring complex protein of the CCT6 subunit of the yeast TRiC complex, a common unsupported membrane-free holey carbon grid was used for frozen sample preparation. From the original cryo-electron microscopy image, almost no target protein particles were observed (Figure 8A). When a grid covered with an unmodified graphene oxide film was used for frozen sample preparation, a very small amount of target protein was visible (Figure 8B). However, using a pyr-NHS-modified graphene oxide grid for frozen sample preparation, efficient enrichment of the target protein can be achieved (Figure 8C).

[0072] The above results show that the affinity grid prepared by the pyr-NHS-modified carbon support membrane grid is of great significance for the cryo-sampling of protein complexes with extremely low abundance. The present invention provides strong support for the cryo-electron microscopy sample preparation process of protein complexes with precious sources, difficult purification, and extremely low expression levels. The use of pyr-NHS-modified carbon support membrane grids for cryo-sampling can achieve the enrichment of the target protein complex and greatly improve the efficiency of cryo-electron microscopy data collection. At the same time, the use of pyr-NHS to modify the carbon support membrane grid is a simple and convenient process that is user-friendly and efficient, and can be used even in non-professional laboratories.

[0073] Example 6: M2 antibody was anchored to the graphene oxide grid surface via pyr-NHS to prepare an affinity grid for affinity enrichment of target protein with FLAG tag

[0074] The affinity grid was prepared by anchoring the FLAG-tagged antibody protein M2 on the surface of the graphene oxide grid via pyr-NHS. The graphene oxide grid modified with pyr-NHS can be used to anchor the antibody protein. The antibody protein used in this example is M2, which can specifically bind to the target protein expressed with a FLAG tag. The specific steps are as follows: (1) Dilute the M2 antibody protein to a concentration of 40 μg / mL using PBS buffer; (2) Take 10 μL of the M2 antibody solution and add it dropwise to the surface of the graphene oxide grid modified with pyr-NHS, and incubate at room temperature for 40 minutes; (3) Use protein buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT) to wash the grid surface to terminate the cross-linking reaction of pyr-NHS with the M2 antibody and remove excess M2 antibody. This cleaning step uses a hydrophobic parafilm loaded with protein buffer. 30 μL of protein buffer is dripped onto the parafilm. The side of the grid anchored with the M2 antibody is briefly placed in contact with the droplet surface. The droplet is then blotted with filter paper along the edge of the grid. This cleaning step is repeated three times to prepare the affinity grid. The antibody protein concentration range is 25-100 μg / mL.

[0075] Affinity grids were used for specific affinity binding and cryopreservation of the purified CCT6 subunit FLAG-tagged TRiC complex target protein. The procedure was as follows: 3 μL of protein solution was added to the prepared affinity grid and incubated at room temperature (~25°C) for 2 minutes. After incubation, the affinity grid was removed from the metal holder using forceps for cryopreservation and the grid surface was washed with protein buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT). This wash process also used a hydrophobic parafilm-coated buffer. 30 μL of protein buffer was added dropwise to the parafilm. The surface of the grid, which had affinity with the target protein CCT6-FLAG TRiC complex, was briefly placed in contact with the buffer droplet. The droplet was then blotted off along the edge of the grid with filter paper. This wash step was repeated three times. Subsequently, the grid with affinity for the CCT6-FLAG TRiC protein was directly subjected to typical cryo-EM sample preparation: taking Vitrobot Mark IV as an example, 2.5 μL of protein buffer was dripped onto the surface of the affinity grid anchored with the target protein, the excess liquid was aspirated using Vitrobot, and then the grid was rapidly frozen using liquid ethane cooled by liquid nitrogen for subsequent cryo-EM data collection and three-dimensional structure analysis.

[0076] Cryo-electron microscopy was used to compare the untreated graphene oxide grid and the affinity grid. Figure 9 shows a cryo-electron microscopy photograph of the sample prepared by freezing after the M2 antibody was anchored to the surface of the graphene oxide grid via pyr-NHS and affinity-bound to the target protein CCT6-FLAG TRiC complex, as provided in an embodiment of the present invention, and its comparative experimental data. It was found that the protein concentration of the untreated graphene oxide grid was low after direct incubation with the target protein solution (Figure 9A); while the affinity grid prepared using the graphene oxide grid had a significant affinity enrichment effect on the target protein CCT6-FLAG TRiC (Figure 9B), which can be used for subsequent cryo-electron microscopy data collection and high-resolution structure analysis.

[0077] 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 various embodiments of the present invention. Industrial Applicability

[0078] The carbonaceous support membrane affinity grid modified with the organic small molecule pyr-NHS in this invention is highly efficient in enriching low-abundance target proteins. Preferably, the organic small molecule pyr-NHS is used to anchor specific antibody proteins to the grid surface. The resulting affinity grid enables efficient affinity purification of target proteins fused to specific tags directly from cell lysates, enabling direct cryo-sample preparation and high-resolution cryo-electron microscopy structural analysis. This reduces sample purification time and steps, improves sample integrity, reduces sample volume requirements, and overcomes the issue of sample distribution bias.

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 proteins include NZ-1 antibody protein, anti-FLAG tag M2 antibody protein and HA tag antibody, HIS tag antibody, and anti-GFP antibody.

3. The affinity grid for cryo-electron microscopy according to claim 1 or 2, characterized in that The affinity carrier net is prepared by a method comprising the following steps: covering the surface of the carbonaceous carrier film on the carrier net with an organic small molecule pyr-NHS to prepare the affinity carrier net; Preferably, the organic small molecule pyr-NHS is used to anchor the specific antibody protein on the surface of the carbonaceous support film covering the support grid.

4. An affinity grid for cryo-electron microscopy, characterized in that: It comprises a carbonaceous carrier film covered on a carrier mesh, and an organic small molecule pyr-NHS connected to the carbonaceous carrier film; the carbonaceous carrier film is a graphene film, an oxidized graphene film or a carbon film.

5. A method for preparing an affinity grid for cryo-electron microscopy, characterized in that: The method comprises the following steps: covering the surface of the carbonaceous carrier film on the carrier net with an organic small molecule pyr-NHS to prepare an affinity carrier net; Preferably, the affinity grid is prepared by anchoring the specific antibody protein on the surface of a carbonaceous carrier film covering the grid using an organic small molecule pyr-NHS; the carbonaceous carrier film is a graphene film, a graphene oxide film or a carbon film.

6. The method for preparing an affinity grid for cryo-electron microscopy according to claim 5, 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, a specific antibody solution is coated on the surface of the grid and incubated to obtain an affinity grid.

7. Use of the affinity grid for cryo-electron microscopy described in any one of claims 1 to 4 or the affinity grid obtained by the method for preparing the affinity grid for cryo-electron microscopy described in claim 5 or 6 in preparing frozen samples for cryo-electron microscopy; preferably, the affinity grid is used for direct and specific affinity and enrichment of target proteins from cell lysates.

8. A method for preparing a frozen sample, characterized in that: include: The organic small molecule pyr-NHS is covered on the surface of the carbonaceous supporting film on the carrier grid, preferably the organic small molecule pyr-NHS is used to anchor the specific antibody protein on the surface of the carbonaceous supporting film covering the carrier grid to prepare an affinity carrier grid; the affinity carrier grid is incubated with a cell lysate containing the target protein, so that the affinity carrier grid 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 a specific expression tag, and the carbonaceous supporting film is a graphene film, a graphene oxide film or a membrane.

9. The method for preparing a frozen sample according to claim 8, 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 a specific expression 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.

10. Use of the frozen sample obtained by the method for preparing a frozen sample according to claim 8 or 9 in the analysis of the structure of biological macromolecules, wherein the application is any of the following: 1) Avoid damage to biomacromolecules caused by the gas-liquid interface during sample preparation; 2) Enrichment of low-abundance target proteins; 3) Overcoming the orientation tendency problem of biological sample distribution; 4) Achieve affinity of target protein directly from cell lysate for frozen sample preparation; 5) Collect cryo-EM data and conduct high-resolution cryo-EM structure analysis.

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