Freeze electron microscope aberration processing method and device, electronic equipment and storage medium

Abstract

The application provides a processing method and device for cryo-EM parallax, an electronic device and a storage medium. The method comprises: identifying a support net image to obtain first position information of target holes; grouping the target holes according to the first position information by using a preset rule to obtain a plurality of grid groups; determining a first grid group from the grid groups; correcting the first position information of the target holes in other grid groups by using the relative position information of the target holes in the first grid group to obtain second position information of the target holes in the other grid groups; photographing based on the second position information by using cryo-EM to obtain grid images corresponding to each target hole; dividing each grid image to different position groups based on the grid groups according to the second position information; and dividing the particles corresponding to the grid images corresponding to the positions of each grid group to the same optical group according to the position group information, so that the particles in the same optical group can be uniformly corrected based on the same parallax.

Description

TECHNICAL FIELD

[0001] Embodiments of the present application relate to the technical field of cryo-EM, in particular to a processing method and device for cryo-EM astigmatism, electronic equipment and storage medium. BACKGROUND

[0002] Cryo-EM is mainly applied in the field of structural biology, and provides high-resolution structures for proteins that are not suitable for crystallization, and explains the relationship between structure and function at the atomic level. In recent years, the method of collecting data based on electron beam deflection has replaced the method of collecting data by mechanical movement of the sample stage, greatly accelerating the efficiency of cryo-EM data collection. In this scheme, when collecting data, the sample stage is mechanically moved from the center of one group to the center of another group, and data is collected at the center point. Then, the sample stage remains stationary, and the electron beam is sequentially deflected to collect data for other points in the group, thereby improving the data collection speed. However, this method introduces additional astigmatism, which reduces the resolution of the reconstruction. After data collection, data processing is performed in relion (or other cryo-EM processing software). During data processing, data collected in the same batch is placed in the same optical group for correction in the subsequent processing process. Due to the difference between the true value and the predicted value of the astigmatism introduced at different positions of the hole, when correcting in the structure analysis based on the same optical group during data processing, it is not possible to accurately correct different astigmatism at different positions, and when mixed together for astigmatism correction, it will result in a low resolution of the three-dimensional structure obtained after image reconstruction processing. SUMMARY

[0003] Embodiments of the present application provide a processing method for cryo-EM astigmatism, comprising:

[0004] identifying a carrier web image to obtain first position information of target hole positions on the carrier web;

[0005] grouping all target hole positions according to the first position information using a preset rule to obtain a plurality of grid groups;

[0006] determining a first grid group from the grid groups, and correcting the first position information of the target hole positions in other grid groups using the relative position information of the target hole positions in the first grid group to obtain second position information of the target hole positions in the other grid groups;

[0007] photographing based on the second position information of the target hole positions in the grid groups using cryo-EM to obtain grid images corresponding to each of the target hole positions;

[0008] The grid images are divided into different position groups according to the second position information based on the grid groups, and the particles corresponding to the grid images at the corresponding positions of each grid group are divided into the same optical group according to the position group information, so that the particles in the same optical group can be uniformly corrected based on the same coma during image processing.

[0009] In some embodiments, the preset rules include:

[0010] The adjacent hole spacing of the target hole position is determined, and the adjacent hole average azimuth is determined based on the adjacent hole spacing.

[0011] A reference hole is determined according to the first position information, and a first boundary line and a second boundary line are determined based on the reference hole and the adjacent hole average azimuth.

[0012] The distances of each hole to the first boundary line and the second boundary line are determined, and all target hole positions are grouped according to the preset size information of the grid group and the distances of each hole to the first boundary line and the second boundary line.

[0013] In some embodiments, the adjacent hole average azimuth is determined based on the adjacent hole spacing, including:

[0014] The adjacent hole average spacing is determined according to the adjacent hole spacing, and the distance standard deviation is determined according to the adjacent hole spacing and the adjacent hole average spacing.

[0015] All target hole positions are screened based on the adjacent hole average spacing and the distance standard deviation, and the target hole positions whose adjacent hole spacing is less than the sum of the adjacent hole average spacing and the distance standard deviation and greater than the difference between the adjacent hole average spacing and the distance standard deviation are screened.

[0016] The adjacent hole azimuths of the screened target hole positions are determined in sequence, and the adjacent hole average azimuth is determined according to all adjacent hole azimuths.

[0017] In some embodiments, the reference hole is determined according to the first position information, including:

[0018] The average position information is determined according to the first position information of all target hole positions, and the closest target hole position is selected as the reference hole according to the average position information.

[0019] In some embodiments, the first position information of the target hole positions in other grid groups is corrected based on the relative position information of the target hole positions in the first grid group to obtain the second position information of the target hole positions in other grid groups, including:

[0020] The relative position relationship between each target hole position and the center hole in the first grid group is traversed to obtain the relative position information.

[0021] The other grid groups are sequentially traversed, the prediction position information of the target hole positions of each other grid group is determined based on the relative position information, the first position information and the prediction position information of each target hole position are compared, and when the comparison result meets a condition, the first position information is updated by using the prediction position information to obtain the second position information.

[0022] In some embodiments, the step of dividing each of the grid images into different position groups according to the second position information comprises:

[0023] The distance and angle of the center hole in the grid group to each other target hole position in the grid group are determined.

[0024] Each other target hole position is separated by using a bisector passing through the center hole according to the angle, so as to divide the grid images corresponding to each target hole position in the grid group into different position groups.

[0025] In another aspect, the embodiments of the present application also provide a processing device for cryo-EM coma, comprising:

[0026] A recognition module is configured to recognize a grid image to obtain first position information of target hole positions on the grid.

[0027] A first grouping module is configured to group all target hole positions according to the first position information by using a preset rule to obtain a plurality of grid groups.

[0028] A correction module is configured to determine a first grid group from the grid groups, and correct the first position information of the target hole positions in other grid groups by using the relative position information of the target hole positions in the first grid group to obtain second position information of the target hole positions in the other grid groups.

[0029] A shooting module is configured to sequentially shoot the target hole positions based on the grid groups by using a cryo-EM to obtain grid images corresponding to each of the target hole positions.

[0030] A data processing module is configured to divide each of the grid images into different position groups according to the second position information based on the grid groups, and divide the particles corresponding to the grid images corresponding to each grid group into a same optical group based on the position group information, so that the particles in the same optical group can be uniformly corrected based on the same coma during image processing.

[0031] In some embodiments, the correction module comprises:

[0032] An obtaining unit is configured to traverse the relative position relationship between each target hole position and the center hole in the first grid group to obtain the relative position information.

[0033] The comparison unit is configured to sequentially traverse the other grid groups, determine predicted position information of target holes of each other grid group based on the relative position information, compare the first position information and the predicted position information of each target hole, and update the first position information with the predicted position information to obtain the second position information when the comparison result meets a condition.

[0034] The electronic device includes at least a memory, a processor and a bus. The memory stores machine readable instructions executable by the processor. When the electronic device is running, the processor communicates with the memory through the bus. When the machine readable instructions are executed by the processor, the processing method of claims 1-6 is implemented.

[0035] The electronic device includes at least a memory, a processor and a bus. The memory stores machine readable instructions executable by the processor. When the electronic device is running, the processor communicates with the memory through the bus. When the machine readable instructions are executed by the processor, the processing method of claims 1-6 is implemented.

[0036] Based on the disclosure of the above embodiments, the beneficial effects of the present embodiments include:

[0037] In the present embodiment, before the cryo-EM collects images using electron beam deflection to introduce coma, the holes are selected through the two-dimensional image of the support grid, and the selected grid holes are grouped and optimized in position information using a preset rule. Then, the cryo-EM collects data for each grid hole using electron beam deflection in units of grid groups, to obtain grid images corresponding to each target hole. After data collection is completed, each grid image corresponding to a grid group is divided into different position groups according to the optimized position information, and the corresponding particles in each grid image are divided into the same optical group according to the position group information of the grid image. The electron beam deflection of the same optical group is consistent, so as to ensure that the coma introduced by the particles in the same optical group is completely consistent, and the coma can be accurately calculated and uniformly corrected at the deflection angle corresponding to the optical group using the electron beam. Therefore, different optical groups can accurately correct different coma, and the resolution of the reconstructed three-dimensional structure image can be significantly improved. BRIEF DESCRIPTION OF DRAWINGS

[0038] In the drawings, which are not necessarily drawn to scale, like numerals can describe similar components in different views. Like numerals having different letter suffixes can represent different instances of similar components. The drawings illustrate generally, by way of example, various embodiments discussed herein and are not intended to limit the disclosure to the embodiments depicted. The same reference numerals in different drawings can identify the same or similar elements. Such embodiments are examples only, and are non-limiting unless otherwise expressly stated, and some embodiments can not include all of the features shown and described herein.

[0039] Figure 1 This is a flowchart of a method for processing cryo-electron microscopy coma according to an embodiment of this application.

[0040] Figure 2 This is a schematic diagram illustrating the calculation of the azimuth angle of adjacent holes in an embodiment of this application;

[0041] Figure 3 This is a schematic diagram of the method for processing cryo-electron microscopy coma in the embodiment of this application during the grouping process of target well sites;

[0042] Figure 4 and Figure 5 These are schematic diagrams of the target hole positions in all grid groups before and after the position information of the target hole positions is corrected in the embodiments of this application;

[0043] Figure 6 This is a schematic diagram illustrating the grouping of grid images in an embodiment of this application;

[0044] Figure 7 This is a schematic diagram showing the grouping of particles at corresponding positions in all grid images in an embodiment of this application;

[0045] Figure 8 This is a structural block diagram of the processing device for cryo-electron microscopy coma in an embodiment of this application;

[0046] Figure 9 This is a schematic diagram of the structure of an electronic device according to an embodiment of this application. Detailed Implementation

[0047] The specific embodiments of this application will now be described in detail with reference to the accompanying drawings, but these are not intended to limit the scope of this application.

[0048] It should be understood that various modifications can be made to the embodiments disclosed herein. Therefore, the following description should not be considered as limiting, but merely as an example of embodiments. Other modifications within the scope and spirit of this application will be apparent to those skilled in the art.

[0049] The accompanying drawings, which are included in and form part of this specification, illustrate embodiments of the present application and, together with the general description of the present application given above and the detailed description of the embodiments given below, serve to explain the principles of the present application.

[0050] These and other features of this application will become apparent from the following description of preferred forms of embodiments given as non-limiting examples, with reference to the accompanying drawings.

[0051] It should also be understood that, while the present application has been described above with reference to specific embodiments, the application is open to many other equivalent forms as would be apparent to persons with ordinary skill in the art, having the benefit of this disclosure. It is therefore not intended that the application be limited, for it will be understood that many modifications and variations thereof can be effected, while remaining within the scope and spirit of the following claims.

[0052] The above and other aspects, features and advantages of the present application will become more apparent from the following detailed description, taken in conjunction with the accompanying drawings, when properly considered together.

[0053] Specific embodiments of the present application are described herein with reference to the accompanying drawings. However, the disclosed embodiments are merely examples of the present application, which can be embodied in many other forms. Well-known and / or repetitive functions and structures are not described in detail in order to avoid obscuring the present application. Therefore, specific structural and functional details disclosed herein are not intended to limit the present application, but merely as a basis for the claims and a representative basis for teaching one skilled in the art to variously employ the present application in virtually any appropriate detailed structure.

[0054] The specification can use phrases such as "in one embodiment", "in another embodiment", "in yet another embodiment", or "in other embodiments", which can refer to one or more of the same or different embodiments under the present application.

[0055] At present, when a set of images of a mesh is taken by cryo-EM, after the data of the center position is collected by the electron beam perpendicular to the center position, the electron beam is then offset in turn to collect the data of other points of the set. The offset angle of the electron beam at each point is different, thus introducing different coma. After the data collection is completed, all the image data collected in the same batch is put into the same optical group for subsequent coma correction, and the correction result obtained is an average correction value. Different comas cannot be corrected, which reduces the resolution of the reconstructed three-dimensional structure image.

[0056] Thus, in the scheme of the embodiments of the present application, before collecting images by cryo-EM, the target hole sites are selected by the two-dimensional image of the support grid, and the selected target hole sites are grouped and optimized in position information by using a preset rule. Then, the data of the target hole sites in each grid group is collected by cryo-EM, and the grid images corresponding to the target hole sites are obtained. After the data collection is completed, each grid image corresponding to the collected data based on one grid group is divided into different position groups according to the optimized position information, and then the corresponding particles in each grid image are divided into the same optical group according to the position group information of the grid image. The electron beam deflection of the same optical group is consistent, so as to ensure that the coma introduced by the particles in the same optical group is completely consistent, and the coma can be accurately calculated and uniformly corrected by using the deflection angle corresponding to the optical group. Thus, different optical groups can perform corresponding accurate correction according to different coma, and the resolution of the reconstructed three-dimensional structure image can be obviously improved.

[0057] Next, the embodiments of the present application will be described in detail in combination with the drawings.

[0058] Figure 1 The flowchart of the processing method of the coma of the electron beam of the cryo-EM according to the embodiments of the present application is shown. As shown in Figure 1 The processing method provided by the embodiments of the present application includes the following steps S1-S5:

[0059] S1, the first position information of the target hole site on the support grid is obtained by recognizing the support grid image;

[0060] In the present application, the support net is used to carry the sample to be detected, so that the whole support net can be imaged by cryo-EM after rapid freezing, and the image containing the whole support net is obtained, that is, the support net image. Of course, the images of each group can also be taken based on the preliminary grouping by cryo-EM, and finally combined to obtain the support net image. Since the plurality of hole sites are uniformly formed on the support net, the sample to be detected, such as biological sample protein, virus, etc., can be carried, and the cryo-EM can obtain the two-dimensional structure image of the biological sample, such as the structure of protein and virus, by imaging in the hole. Therefore, in this step, in order to select the required hole site on the support net for imaging, the support net image is first identified and processed to eliminate unqualified hole sites, such as empty hole sites not carrying biological samples, or hole sites with broken biological samples, etc., and based on the detection requirements, each hole site meeting the conditions on the support net is determined as a target hole site, so that subsequent imaging of the biological sample by cryo-EM can be performed on the target hole site meeting the requirements. Here, the detection requirements can be determined according to the settings of the technician, such as the technician specifying the radius parameter or the gray value, etc., which are not limited in the present application. After obtaining each target hole site, the corresponding coordinate information is recorded to obtain the first position information for subsequent grouping processing of each target hole site. In some specific applications, the software can identify and select the hole site based on the set parameters to determine the target hole site and record the coordinate information of the target hole site.

[0061] For example, the automatic hole selection software can be used to identify the support net image based on the set parameter conditions to determine the target hole site and obtain the coordinate information of the target hole site on the support net to obtain the first position information of the target hole site on the support net. Specifically, the automatic hole selection process can be:

[0062] The images taken by cryo-EM based on the preliminary grouping are exported as jpg files by SerialEM and saved in a folder;

[0063] The picture position, nav file and other parameters are set in the automatic hole selection software in sequence, and the run button is clicked;

[0064] The software uses the Hough circle recognition algorithm and the template matching algorithm to identify the circular holes in the image according to the set conditions, obtains the target hole site, and records the coordinates of the center of the circular hole in the map, and sequentially records all the coordinate positions.

[0065] The images of each group are subjected to the hole selection operation according to the step, and after completion, the target hole site of the whole support net and the corresponding coordinate information are obtained.

[0066] S2, grouping all target hole positions according to the first position information by using a preset rule to obtain a plurality of grid groups.

[0067] This step aims to group each target hole position by using a preset rule, and divide it into a plurality of grid groups of the same size for subsequent processing. In specific applications, the preset rule can be set according to actual needs. In actual applications, the size of the grid group can be set according to the pitch of the grid on the grid to include a corresponding number of grid holes. Here, the grid hole is the actual grid hole set on the grid, so that all target hole positions can be grouped based on the size of the required grid group by using the corresponding preset rule. For example, the technician can set the size of the grid group to 3*3, 4*4, 5*5, etc. according to the needs of the actual shooting process, wherein 3*3 means that the grid group is 3 hole distances in the length direction and 3 hole distances in the width direction, and the grid group includes 9 grid holes, all or part of which are the required target hole positions.

[0068] In order to group the target hole positions, the preset rule includes:

[0069] determining the adjacent hole distance of the target hole position, and determining the average azimuth angle of the adjacent hole based on the adjacent hole distance;

[0070] determining a reference hole according to the first position information, and determining a first boundary line and a second boundary line based on the reference hole and the average azimuth angle of the adjacent hole;

[0071] determining the distance of each hole to the first boundary line and the second boundary line, and grouping all target hole positions according to the preset size information of the grid group and the distance of each hole to the first boundary line and the second boundary line.

[0072] In some specific applications, the process of implementing the preset rule for grouping includes the following steps A1-A7:

[0073] A1, set the size of the grid group as WidthNumxLengthNum, and the following calculation is illustrated by taking 3x3 as an example.

[0074] A2, calculate the adjacent hole distance d ij , that is, the distance from each target hole position (x i , y i ) to the nearest distance target hole position (x j , y j ), the distance of the nearest hole is calculated using the kd-tree algorithm, and the distance d ij from all target hole positions to the nearest distance target hole position is recorded, and the average distance is calculated according to the adjacent hole distance d ij to obtain the average adjacent hole distance Again according to the adjacent hole spacing d ij And the average spacing of adjacent holes Calculate the distance standard deviation sd ij .

[0075] A3, based on the adjacent hole spacing d ij Determine the average azimuth angle of adjacent holes

[0076] A3.1, first filter out the target hole position whose adjacent hole spacing d ij is less than the sum of the average spacing of adjacent holes and the distance standard deviation (i.e. ) and greater than the difference between the average spacing of adjacent holes and the distance standard deviation .

[0077] A3.2, based on the filtered target hole position, calculate the azimuth angle of each target hole position (x i , y i ) to the nearest distance target hole position (x j , y j ) in turn and convert it to (0-90°), denoted as the adjacent hole azimuth angle θ ij .

[0078] In specific implementation, the angle μ (the angle from one point to the other point counterclockwise from the x-axis) of the two nearest target hole positions on the same carrier network is calculated in turn. Assuming that the coordinates of the two points are P1(x1, y1) and P2(x2, y2), the Python example code is as follows:

[0079] import math

[0080] import cmath

[0081] angle = math.degrees(cmath.polar(complex(x2-x1, y2-y1))[-1])

[0082] if angle < 0:

[0083] angle = 360 + angle

[0084] Then convert the value of angle μ to an angle less than 90°, and the conversion method is as follows: first, determine whether μ is greater than 180°, and update the value of μ μ = μ-180° when μ is greater than 180°; then determine whether μ is greater than 90°, and update the value of μ μ = μ-90° when μ is greater than 90°.

[0085] Combined with Figure 2The angle of B point relative to A point is 63.43°. The angle of C point relative to A point is 153.43°, and since the value is greater than 90°, 153.43°-90°=63.43°. The angle of A point relative to C point is 333.43°, and since the value is greater than 180°, the angle is updated as 333.43°-90°=153.43°, and since 153.43°>90°, the angle is updated as 153.43°-90°=63.43°.

[0086] A3.3, based on the adjacent azimuth angle θ of the screened target hole site ij Calculate the average value to obtain the average azimuth angle of adjacent holes

[0087] A4, determine the reference hole. The reference hole can be selected and determined according to actual needs. Here, in order to facilitate calculation, the center hole of all target hole sites can be taken as the reference hole for calculation. When determining the center hole, first calculate the average coordinate position of all target hole sites Then calculate the hole closest to position A as the center hole O(x0, y0), see, for example Figure 3 .

[0088] A5, draw a straight line l1 passing through 0 point and having an angle of with the x axis, draw a straight line l2 passing through A point and perpendicular to l1, and respectively calculate the equations of the two straight lines, as follows:

[0089] l1: A1x+B1y+C1=0

[0090] l2: A2x+B2y+C2=0

[0091] A6, calculate the distance m and m of the center C(x , y ) of each target hole site to the two straight lines l1 and l2 in turn (including positive and negative to indicate which side of the straight line), and the calculation formula is as follows:

[0092]

[0093]

[0094] A7, group all points, and the grouping formula and calculation method are as follows:

[0095] A7.1, group according to :

[0096] A7.2, group according to :

[0097] In the above grouping formula, int(·) represents rounding the data, abs(·) represents taking the absolute value of the data, approximately equal to the actual distance of the adjacent mesh on the grid.

[0098] A7.3, according to the calculation results of steps A7.1 and A7.2 and determine the grouping of the target hole, and calculate The values of different target holes are equal, and The values of different target holes are equal, and Figure 3 In the above grouping formula, int(·) represents rounding the data, abs(·) represents taking the absolute value of the data, In some actual applications, the center hole in each grid group can be determined and recorded for subsequent use. When determining the center hole of the grid group, first calculate the average coordinate position of all target holes in the grid group

[0099] Then calculate the hole closest to position B in the grid group as the center hole. In some specific implementation processes, the target holes in the grid group can be numbered and named for ease of recording and use. For example, the number of the center hole in the group can be set to 1, and the name can be named in combination with the group name. The center hole of the grid group can be named as "201-1", indicating that the center hole is the first hole of group number 201, i.e. the center hole, and then the other holes in the group are numbered, and the corresponding are "201-2", "201-3", etc. Of course, the numbering can also be performed according to the actual use of the technician, which is not limited herein.

[0100] S3, determining a first grid group from the grid groups, correcting first position information of target holes in other grid groups with relative position information of target holes in the first grid group to obtain second position information of target holes in the other grid groups.

[0101] In this step, based on the grid groups obtained by grouping, a first grid group is determined as a template group, and the position of holes in other grid groups is optimized using the template group so that the relative position relationship of the holes in the other grid groups is the same as that of the template group. In some specific applications, the first grid group needs to be selected from the grid groups in which the target holes are relatively complete. The selection can be made manually by observation or automatically by a program. For example, in the case of 3*3 grouping, there are 9 holes in each position in the grid group. A technician can determine that the first target hole in the 9th grid group is the first grid group, and the position information of the target holes in other grid groups is optimized and corrected.

[0102] In some embodiments, the correction of the first position information of the target holes in the other grid groups with the relative position information of the target holes in the first grid group to obtain the second position information of the target holes in the other grid groups includes:

[0103] S31, obtaining the relative position information by traversing the relative position relationship between each target hole and the center hole in the first grid group;

[0104] S32, sequentially traversing the other grid groups, determining the predicted position information of the target holes in each other grid group based on the relative position information, comparing the first position information and the predicted position information of each target hole, and when the comparison result meets the condition, updating the first position information with the predicted position information to obtain the second position information.

[0105] In step S31, when calculating the relative position relationship of each target hole in the first grid group, the relative position relationship between each target hole and the center hole in the first grid group is sequentially calculated. For example, in a 3*3 grid, the relative position relationship formula in step S31 is as follows:

[0106]

[0107] In step S32, the target hole coordinates of all other grid groups are optimized and corrected by sequentially traversing the other grid groups.

[0108] For example, for grid group A, the predicted position coordinates (x n_predict , y n_preditct ) of all target holes in grid group A are calculated sequentially according to the relative position relationship formula in step S31.

[0109] The distance between the hole site coordinate and the predicted position coordinate is calculated, and when calculating, the center hole in the grid group A can be taken as the basis, and the relative position formula is combined to calculate. If there is a predicted position coordinate and a hole site coordinate distance less than d min , then the coordinate information of the hole site is replaced with the predicted coordinate value, if there is no predicted position and hole site coordinate distance less than d min , then delete this hole. In this embodiment, the maximum value of d min is the radius of the hole, and the smaller the value, the more stringent the standard.

[0110] Figure 4 and Figure 5 are respectively the schematic diagram of the target hole site of all grid groups before and after the position information of the target hole site is corrected in the embodiment of the application. As shown in Figure 4 , when all grid groups are combined together with the center hole coordinate as (0, 0), due to the accuracy problem of punching on the grid, the position relationship of the corresponding hole sites between the grid groups is not completely the same, that is, the coordinate information data of the target hole site on the corresponding position of each grid group cannot completely coincide, and there is a certain error. When the cryo-EM is based on the coordinate information of the target hole site for shooting, the deflection angle of the electron beam is also different, and the introduced coma is also different. After the scheme of the embodiment is corrected, it can be seen from Figure 5 that the corresponding position relationship between each grid group is the same, and the coordinate information data of the target hole site on the corresponding position of each grid group completely coincides, so that the coma introduced by the data collected by the electron beam deflection in the same direction is completely the same, so as to facilitate the accurate correction of the coma subsequently.

[0111] In some actual applications, the optimized coordinates of the target hole site in each grid group can be saved to the coordinate file that SerialEM can recognize, and the file name is file_nav.nav, so as to facilitate subsequent data collection.

[0112] S4, using cryo-EM to shoot based on the second position information of the target hole site in the grid group, to obtain the grid image corresponding to each target hole site.

[0113] In this step, the cryo-EM performs image acquisition in grid groups as units, and obtains grid images corresponding to each target hole position. Specifically, the cryo-EM performs image acquisition in grid groups as units, collects image data of the central hole by using the electron beam perpendicular to the central position, and obtains the grid image corresponding to the central hole; and then the electron beam is offset in sequence to the image data of other target hole positions of the grid group, so as to obtain the grid images corresponding to all target hole positions in the grid group. In some practical applications, the software SerialEM is used to collect data, and the coordinate file saved after the coordinate information of the target hole position is corrected in step S3 is imported by using the open source SerialEM software. The machine is adjusted and the data of the target hole position is collected. In the data collection process, the number of the hole and the file name (such as tif file) obtained by collection are recorded into the log file.

[0114] Then the log file is textually parsed to obtain the corresponding relationship between the number of the target hole position and the tif file during shooting, and the data is saved to the database or other storage file file_log, so as to facilitate subsequent processing.

[0115] In this application, the software Relion3.1 and above versions can also be used to perform a series of preprocessing steps on the grid image data to obtain preliminary processing results, including Motion Correction, CTF, particle selection, Class2D, Class3D, Refine3D to obtain preprocessed results, so as to facilitate subsequent image processing.

[0116] S5, based on the second position information, each grid image is divided into different position groups, and the particles corresponding to the grid images corresponding to the position of each grid group are divided into the same optical group according to the position group information, so that the particles in the same optical group can be uniformly corrected based on the same coma during image processing.

[0117] In this step, considering that the deflection angle of the electron beam is different when collecting each grid image, different coma will be introduced, therefore, first, according to the second position information, the position groups of each grid image are divided, and the second position information is the position information after position optimization and correction based on the first grid group, so that after grouping, the position group information of each grid image can reflect the corresponding position information in the grid group, and then according to the position group information, the grid images corresponding to the position of the grid group are divided into the same kind, so that the particles corresponding to the same group of grid images can be divided into the same optical group. After grouping processing, the deflection of the electron beam of the same optical group during image acquisition is consistent, so that the coma introduced by the particles in the same optical group is completely consistent, so that the particles in the same optical group can be corrected based on the same coma during image processing, and the accuracy during data correction is improved.

[0118] In some embodiments, the dividing of each of the grid images into different position groups according to the second position information comprises:

[0119] determining the distance and angle of the center hole in the grid group to each other target hole position in the grid group;

[0120] dividing each of the other target hole positions according to the angle using the bisector of the center hole to divide the grid images corresponding to each target hole position in the grid group into different position groups.

[0121] In some specific implementations, the distance and angle of the other target hole positions in the grid group to the center hole can be calculated based on the second position information and angle Then divide into τ groups according to the distance from the center hole. For example, a 3x3 group can be divided into 2 groups, with the center point as a group G d1 , and the other points as a group G d2 .

[0122] The above G d2 group is further divided into groups according to the corresponding angle, to ensure that points of different angles are divided into different groups. For example, as shown in Figure 6 , the G d2 group has 8 positions with different angles, so the target hole positions corresponding to the 8 positions need to be divided into 8 groups, and the plane coordinate system with the center group as the origin needs to be divided into at least 8 parts according to the angle, each part is 45°; different groups are named according to the angle , numbered from 1-9, of course, the numbering can also be according to actual needs. Thus, the grid images corresponding to the target hole positions can be divided into corresponding position groups based on the grouping of the target hole positions, so as to represent the target hole positions photographed by the grid images through the position group information.

[0123] In some practical applications, the corresponding particle information in the grid image can be obtained by data processing, and then the star file obtained by the relion preprocessing is modified according to the optical group information of the grid image, and the information of data_optics is modified according to the new optical group division.

[0124] The star file obtained by the relion preprocessing is modified, and the optical group information of each particle in data_particles is updated.

[0125] Then, according to the optical group information of each particle, the particles in all grid images corresponding to the angle in the grid group are divided into the same optical group, and the particles in the same optical group can be accurately corrected based on the same coma. For example, as shown in Figure 7 For example, when the grid group is 3x3, the corresponding particles in the grid images corresponding to the angle in all grid groups have the same optical group information, which can be divided into 9 optical groups, and different optical groups are accurately corrected by different coma, so as to improve the resolution of the three-dimensional structure image obtained after image reconstruction processing.

[0126] Based on the same inventive concept, the embodiment of the present application also provides a processing device for cryo-EM coma, Figure 8 The block diagram of the processing device provided by the embodiment of the present application is shown in Figure 8 The processing device comprises:

[0127] The identification module 10 is configured to identify the grid image to obtain the first position information of the target hole position on the grid;

[0128] The first grouping module 20 is configured to group all target hole positions according to the first position information by using a preset rule to obtain a plurality of grid groups;

[0129] The correction module 30 is configured to determine a first grid group from the grid groups, and correct the first position information of the target hole positions in other grid groups by using the relative position information of the target hole positions in the first grid group to obtain the second position information of the target hole positions in other grid groups;

[0130] The shooting module 40 is configured to shoot the target hole positions based on the grid groups by using the cryo-EM to obtain the grid images corresponding to each target hole position;

[0131] The data processing module 50 is configured to divide each grid image into different optical groups according to the second position information based on the grid groups, and divide the particles corresponding to the grid images at the corresponding positions of each grid group into the same optical group according to the position group information, so that the particles in the same optical group can be uniformly corrected based on the same coma during image processing.

[0132] In some embodiments, the correction module comprises:

[0133] The obtaining unit is configured to obtain the relative position information by traversing the relative position relationship between each target hole site and the center hole in the first grid group.

[0134] The comparison unit is configured to traverse other grid groups in sequence, determine the predicted position information of the target hole site of each other grid group based on the relative position information, compare the first position information and the predicted position information of each target hole site, and when the comparison result meets the condition, update the first position information with the predicted position information to obtain the second position information.

[0135] The processing device in the embodiments of the present application can realize the processing method for cryo-EM coma provided in any embodiment of the present application through the function modules thereof.

[0136] The embodiments of the present application further provide an electronic device comprising at least a memory 901, a processor 902 and a bus (not shown), wherein a structural schematic diagram of the electronic device can be as shown in Figure 9 The memory 901 stores machine readable instructions executable by the processor 902, and when the electronic device is running, the processor 902 and the memory 901 communicate through the bus, and the machine readable instructions are executed by the processor to perform the steps of the processing method for cryo-EM coma provided in any embodiment of the present application.

[0137] Since the electronic device introduced in the embodiments of the present application is an electronic device provided with a memory for implementing the processing method for cryo-EM coma disclosed in the embodiments of the present application, based on the processing method introduced in the embodiments of the present application, those skilled in the art can understand the structure and deformation of the electronic device introduced in the embodiments of the present application, and therefore will not be described here.

[0138] The embodiments of the present application further provide a storage medium, which stores one or more programs, and when the one or more programs are executed by a processor, the steps of the processing method for cryo-EM coma provided in any embodiment of the present application are implemented.

[0139] The storage medium in the embodiments can be included in an electronic device / system, or can exist separately and not be assembled into the electronic device / system. The storage medium stores one or more programs, which, when executed, implement the steps of the processing method provided by the embodiments of the present application.

[0140] According to the embodiments of the present application, the computer readable storage medium can be a non-volatile computer readable storage medium, which can include, but is not limited to, a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), a portable compact disc read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any appropriate combination thereof. Alternatively, the specific examples in the embodiments can refer to the examples described in any of the embodiments of the present application, which are not repeated here. Obviously, those skilled in the art should understand that the above-mentioned modules or steps of the present application can be realized by a general computing device, which can be concentrated on a single computing device, or distributed on a network composed of multiple computing devices, and alternatively, they can be realized by program codes executable by a computing device, so that they can be stored in a storage device and executed by a computing device, and in some cases, the steps shown or described can be executed in an order different from that shown here, or they can be manufactured into individual integrated circuit modules, or a plurality of modules or steps can be manufactured into a single integrated circuit module. Thus, the present application is not limited to any specific combination of hardware and software.

[0141] It should be understood that although the present application is described according to various embodiments, not every embodiment contains only one independent technical solution, and the description of the specification is only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.

[0142] The above embodiments are only exemplary embodiments of the present application and are not used to limit the present application, and the protection scope of the present application is defined by the claims. Those skilled in the art can make various modifications or equivalent replacements to the present application within the spirit and protection scope of the present application, and such modifications or equivalent replacements should also be considered to fall within the protection scope of the present application.

Claims

1. A method for processing coma in cryo-electron microscopy, wherein, include: The first position information of the target hole on the net is obtained by recognizing the net image. Using preset rules, all target holes are grouped according to the first location information to obtain multiple grid groups; A first grid group is determined from the grid groups, and the first position information of the target holes in other grid groups is corrected by the relative position information of the target holes in the first grid group to obtain the second position information of the target holes in other grid groups. Cryo-electron microscopy is used to capture images based on the second position information of the target holes within the grid group to obtain grid images corresponding to each target hole. Based on the grid group, each grid image is divided into different position groups according to the second position information. Based on the position group information, the particles corresponding to the grid images at the corresponding positions of each grid group are divided into the same optical group, so that the particles in the same optical group can be uniformly corrected based on the same coma during image processing. in, The step of dividing each of the grid images into different location groups according to the second location information includes: Determine the distance and angle from the center hole within the grid group to each other target hole within the grid group; Based on the aforementioned angle, the other target holes are separated using bisectors passing through the central hole, thereby dividing the grid images corresponding to each target hole within the grid group into different position groups.

2. The method according to claim 1, wherein, Preset rules, including: Determine the spacing between adjacent holes at the target hole location, and determine the average azimuth angle of adjacent holes based on the spacing between adjacent holes; The reference hole is determined based on the first position information, and the first and second boundary lines are determined based on the average azimuth angles of the reference hole and adjacent holes. Determine the distance of each hole to the first and second dividing lines, and group all target hole positions according to the preset size information of the grid group and the distance of each hole to the first and second dividing lines.

3. The method according to claim 2, wherein, The method of determining the average azimuth angle of adjacent holes based on the spacing between adjacent holes includes: The average distance between adjacent holes is determined based on the distance between adjacent holes, and the standard deviation of the distance is determined based on the distance between adjacent holes and the average distance between adjacent holes. All target holes are screened based on the average spacing between adjacent holes and the standard deviation of the distance. Target holes with a spacing between adjacent holes that is less than the sum of the average spacing between adjacent holes and the standard deviation of the distance, and greater than the difference between the average spacing between adjacent holes and the standard deviation of the distance, are selected. Based on the selected target hole positions, the azimuth angles of adjacent holes are determined sequentially, and the average azimuth angle of the adjacent holes is determined based on the azimuth angles of all adjacent holes.

4. The method according to claim 2, wherein, Determining the reference hole based on the first position information includes: The average position information is determined based on the first position information of all target holes, and the target hole closest to the target hole is selected as the reference hole based on the average position information.

5. The method according to claim 1, wherein, The step of correcting the first position information of target holes in other grid groups using the relative position information of target holes in the first grid group to obtain the second position information of target holes in other grid groups includes: The relative position information is obtained by traversing the relative position relationship between each target hole and the center hole within the first grid group; The other grid groups are traversed sequentially. Based on the relative position information, the predicted position information of the target hole in each other grid group is determined. The first position information and the predicted position information of each target hole are compared. When the comparison result meets the conditions, the first position information is updated using the predicted position information to obtain the second position information.

6. A processing device for cryo-electron microscopy coma, wherein, include: The recognition module is used to recognize the netting image and obtain the first position information of the target hole on the netting; The first grouping module is used to group all target holes according to the first location information using preset rules to obtain multiple grid groups; The correction module is used to determine a first grid group from the grid group, and use the relative position information of the target holes in the first grid group to correct the first position information of the target holes in other grid groups, so as to obtain the second position information of the target holes in other grid groups. The imaging module is used to sequentially image the target holes based on the grid group using a cryo-electron microscope to obtain grid images corresponding to each target hole. The data processing module is used to divide each of the grid images into different position groups based on the second position information, and to divide the particles corresponding to the grid images at the corresponding positions of each grid group into the same optical group based on the position group information, so that the particles in the same optical group can be uniformly corrected based on the same coma during image processing. in, The step of dividing each of the grid images into different location groups according to the second location information includes: Determine the distance and angle from the center hole within the grid group to each other target hole within the grid group; Based on the aforementioned angle, the other target holes are separated using bisectors passing through the central hole, thereby dividing the grid images corresponding to each target hole within the grid group into different position groups.

7. The apparatus for processing cryo-electron microscopy coma according to claim 6, wherein, The correction module includes: The acquisition unit is used to traverse the relative position relationship between each target hole and the center hole within the first grid group to obtain the relative position information; The comparison unit is used to sequentially traverse other grid groups, determine the predicted position information of the target hole in each other grid group based on the relative position information, compare the first position information and the predicted position information of each target hole, and when the comparison result meets the conditions, update the first position information using the predicted position information to obtain the second position information.

8. An electronic device comprising at least a memory, a processor, and a bus, wherein the memory stores machine-readable instructions executable by the processor, and when the electronic device is in operation, the processor communicates with the memory via the bus, and when the machine-readable instructions are executed by the processor, the processing method as described in claims 1-5 is implemented.

9. A storage medium storing one or more programs that, when executed by a processor, implement the processing method as described in claims 1-5.

Images