Automated molecular dynamics simulation method and system for enzyme reaction intermediate complex

Abstract

The present disclosure relates to an automated molecular dynamics simulation method and system for an enzyme reaction intermediate complex. The method comprises: obtaining a protein ligand structure file containing a P450 enzyme, parsing to obtain a heme structure file, a common ligand structure file and a pure protein structure file and performing preprocessing; mutating the pure protein structure file based on an acquired mutation setting file and performing a second preprocessing; modifying the pure protein structure file and the heme structure file based on a quantum mechanics QM calculation result file to obtain an intermediate pure protein structure file and an intermediate heme structure file, assembling a molecular dynamics simulation input file based on the intermediate pure protein structure file, the intermediate heme structure file and the common ligand structure file; constructing a molecular dynamics simulation process and generating an automated script; running the automated script to obtain a calculation result file; reading the calculation result file, calculating target parameters and performing visual display.

Description

Technical Field

[0001] The present disclosure relates to the field of biological computing, and in particular, to a method and system for automated molecular dynamics simulation of enzyme reaction intermediate complexes. Background Art

[0002] Cytochrome P450 (CYP) enzymes are a class of superfamily proteins containing heme iron, which widely mediate phase I and phase II metabolism. In the field of medicinal chemistry, P450 enzymes can recognize and catalyze specific compounds. The study of substrate selectivity helps to understand the function of enzymes, predict drug metabolites, evaluate potential drug-drug interactions, and guide safe drug use, which is of great significance to drug design and development. In addition, the transformation of P450 enzymes through synthetic biology methods can further optimize catalytic performance, improve product selectivity and yield, and has important application value in the fields of biomanufacturing, drug synthesis and environmental protection.

[0003] The catalytic mechanism of P450 enzymes is very complex, involving dynamic changes in multiple intermediate states. The structure and properties of these intermediate states directly determine the catalytic efficiency and selectivity. Traditional experimental methods explain the catalytic mechanism to a certain extent, but it is difficult to capture the dynamic process of the intermediate state. For example, X-ray crystallography can provide static enzyme structures, but it cannot capture dynamic changes and predict product selectivity.

[0004] Molecular dynamics simulation (MD) is a tool that can simulate molecular motion at the atomic level. It can reveal the key mechanisms in the catalytic process through the simulation of intermediate states and predict reaction selectivity.

[0005] Although there are several MD software or platforms on the market, they are only applicable to common complex systems. There is a lack of good adaptation for metal ions, more specifically, P450 enzyme intermediates. Therefore, common MD simulation frameworks cannot simulate complexes of P450 enzyme intermediates and cannot reliably explain the mechanism of site selectivity in the catalytic process. Summary of the invention

[0006] The purpose of the present disclosure is to provide an automated molecular dynamics simulation method and system for enzyme reaction intermediate complexes, aiming to meet the experimental needs of synthetic biology and the professional needs of mechanism research by simulating P450 enzyme intermediate complexes.

[0007] In order to achieve the above-mentioned object, the present disclosure provides, in a first aspect, a method for automated molecular dynamics simulation of an enzyme reaction intermediate complex, the method comprising:

[0008] Obtaining an input protein ligand structure file containing a P450 enzyme, parsing the protein ligand structure file to obtain a heme structure file, a common ligand structure file, and a pure protein structure file of the P450 enzyme;

[0009] Preprocess the pure protein structure file, heme structure file and common ligand structure file respectively;

[0010] Obtaining a mutation setting file, mutating a designated site in the preprocessed purified protein structure file based on the mutation setting file, and performing a second preprocessing on the mutated purified protein structure file;

[0011] Based on the preset quantum mechanics QM calculation result file, the pure protein structure file after the second preprocessing and the preprocessed heme structure file are modified to obtain the intermediate pure protein structure file and the intermediate heme structure file respectively, and the molecular dynamics simulation input file containing the intermediate complex structure is assembled based on the intermediate pure protein structure file, the intermediate heme structure file and the preprocessed common ligand structure file;

[0012] Generate constraint files and index files for molecular dynamics simulation, build molecular dynamics simulation process and generate automation scripts;

[0013] Running the automated script to perform a molecular dynamics simulation operation on the input file to obtain an operation result file;

[0014] Read the operation result file, calculate the target parameters and display them visually.

[0015] Optionally, the protein ligand structure file is a protein ligand complex structure file containing a P450 enzyme or a set of files containing a heme structure file, a common ligand structure file and a pure protein structure file of a P450 enzyme, and the parsing of the protein ligand structure file to obtain the heme structure file, the common ligand structure file and the pure protein structure file of the P450 enzyme comprises:

[0016] If the protein-ligand structure file is a protein-ligand complex structure file, the P450 enzyme structure in the protein-ligand complex structure file is identified, and the pure protein structure of the P450 enzyme is retained according to the standard residues and the blocking residues to obtain the pure protein structure file. Based on the HETATM identifier, the heme structure and other ligand structures in the reaction pocket in the protein-ligand complex structure file are identified and extracted to obtain the heme structure file and the common ligand structure file, respectively.

[0017] Optionally, the preprocessing of the pure protein structure file, the heme structure file and the common ligand structure file respectively comprises:

[0018] Protonate the pure protein structure of the pure protein structure file, and reorder the residues of the pure protein structure with disrupted sequence or discontinuity to obtain a pre-processed pure protein structure file;

[0019] Hydrogenate the heme structure file and adjust the atom number to obtain the preprocessed heme structure file;

[0020] The common ligand structure file is neutralized to obtain the preprocessed common ligand structure file, and the structure file and topology file of the ligand molecule are generated based on the preprocessed common ligand structure file, and the corresponding tleap configuration file is generated based on the structure file, and the tleap configuration file is run to obtain the corresponding lib file.

[0021] Optionally, based on the preset quantum mechanics QM calculation result file, modifying the purified protein structure file after the second pretreatment and the pretreated heme structure file to obtain the intermediate purified protein structure file and the intermediate heme structure file respectively includes:

[0022] Based on the preset quantum mechanics QM calculation result file, the hydrogen atom named HG in the 400th CYS residue in the pure protein structure file after the second preprocessing is deleted to match the actual situation of the CPDI state of the QM calculation result file, and the CYS residue with the hydrogen atom deleted is renamed CYM to obtain an intermediate pure protein structure file;

[0023] Based on the preset QM calculation result file, the atomic names in the preprocessed heme structure file are matched, the atomic order in the preprocessed heme structure file is modified according to the atomic order in the QM calculation result file, the O1 atom corresponding to the CPDI state of the QM calculation result file is added to the preprocessed heme structure file, and a connection relationship is established between the O1 atom and the divalent high-spin iron atom in the preprocessed heme structure file to obtain an intermediate heme structure file.

[0024] Optionally, the molecular dynamics simulation input file containing the intermediate complex structure is assembled based on the intermediate pure protein structure file, the intermediate heme structure file and the pre-processed common ligand structure file, and includes:

[0025] Assembling the intermediate pure protein structure file, the intermediate heme structure file and the pre-treated common ligand structure file to obtain the intermediate complex structure file;

[0026] Build the tleap input file of the complex of pure protein, hemoglobin and ligand, obtain the force field of protein, small molecule and water selected by the user and load it using source parameters, use loadamberparams to load the intermediate complex structure file, lib file, structure file and topology file of ligand molecule, use loadpdb to load the preset complex template structure, use addions to automatically add the neutralized ion model of the force field, use slovatebox to load the specified water box model, and finally use saveamberparm to generate the coordinate file and topology file in Amber format as the molecular dynamics simulation input file containing the intermediate complex structure.

[0027] Optionally, the molecular dynamics simulation process includes an energy minimization step, an ensemble balancing step and a production simulation step which are performed sequentially.

[0028] Optionally, the ensemble equilibration step includes an NVE ensemble equilibration step, an NVT ensemble equilibration step and an NPT ensemble equilibration step.

[0029] Optionally, the reading of the operation result file, calculating the target parameters and performing visual display includes:

[0030] Read the calculation result file, use RDKit to label the atomic number of the ligand of the regional selectivity result, and use PyMol to complete the visualization;

[0031] Extract the atomic coordinates in the calculation result file, calculate the distance between the ligand atom and the intermediate heme O1 atom based on the atomic coordinates, and use matplotlib for visualization;

[0032] The minimum distance pair between the ligand atom and the surrounding residues in the calculation results is calculated. The records of interactions during the simulation time are retained according to the distance threshold modified by the user. The interaction between the ligand and the surrounding residues is visualized using matplotlib.

[0033] A second aspect of the present disclosure provides an automated molecular dynamics simulation system for an enzyme reaction intermediate complex, the system comprising:

[0034] A structure file parsing module, used for obtaining an input protein ligand structure file containing a P450 enzyme, parsing the protein ligand structure file to obtain a heme structure file, a common ligand structure file, and a pure protein structure file of the P450 enzyme;

[0035] The structure preprocessing module is used to preprocess the pure protein structure file, heme structure file and common ligand structure file respectively;

[0036] A protein mutation module, used to obtain a mutation setting file, mutate a specified site in the preprocessed pure protein structure file based on the mutation setting file, and perform a second preprocessing on the mutated pure protein structure file;

[0037] The intermediate state preparation module is used to modify the pure protein structure file after the second pretreatment and the heme structure file after the pretreatment based on the preset quantum mechanics QM calculation result file, and obtain the intermediate state pure protein structure file and the intermediate state heme structure file respectively, and assemble the molecular dynamics simulation input file containing the intermediate state complex structure based on the intermediate state pure protein structure file, the intermediate state heme structure file and the pretreated common ligand structure file;

[0038] Simulation building module, used to generate constraint files and index files for molecular dynamics simulation, build molecular dynamics simulation process and generate automation scripts;

[0039] An automated molecular dynamics simulation module, used for running the automated script to perform a molecular dynamics simulation operation on the input file to obtain an operation result file;

[0040] The predefined result analysis module is used to read the operation result file, calculate the target parameters and display them visually.

[0041] A third aspect of the present disclosure provides an electronic device, including:

[0042] a memory having a computer program stored thereon;

[0043] A processor is used to execute the computer program in the memory to implement the steps of any one of the methods described in the first aspect above.

[0044] Through the above technical scheme, after the pure protein structure file, heme structure file and common ligand structure file of the P450 enzyme are parsed, these structure files are preprocessed, mutated and intermediate modified respectively, and finally a molecular dynamics simulation input file containing the intermediate complex structure is assembled based on the intermediate pure protein structure file, the intermediate heme structure file and the preprocessed common ligand structure file, and then a molecular dynamics simulation operation script is prepared, and the input file is subjected to molecular dynamics simulation calculation by running the script to obtain an operation result file, and the target parameters can be calculated and visualized based on the operation result file, thus constructing a set of ligand reaction selectivity prediction processes suitable for P450 enzymes and simulation methods for intermediate complex systems, and completing the MD simulation of the intermediate complex by automation, so that experimental personnel can also perform professional computational simulation predictions, thereby reducing experimental costs.

[0045] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings:

[0047] Figure 1 The present invention is a flowchart of an automated molecular dynamics simulation method for an enzyme reaction intermediate complex according to an exemplary embodiment.

[0048] Figure 2 is another flow chart of a method for automated molecular dynamics simulation of an enzyme reaction intermediate complex according to an exemplary embodiment.

[0049] Figure 3A It is a schematic diagram of the heme-related structure of the intermediate state (CPDI state) of the P450 enzyme according to an exemplary embodiment.

[0050] Figure 3B Schematic diagram of heme-related structural distance parameters in the intermediate state (CPDI state) of a P450 enzyme according to an exemplary embodiment.

[0051] Figure 4 is a diagram showing simulation results of a composite system according to an exemplary embodiment.

[0052] Figure 5A is a schematic diagram showing the differences in ligand regioselectivity sites according to an exemplary embodiment.

[0053] Figure 5B is a schematic diagram showing the distances between different regioselective sites and the O1 atom on the intermediate heme according to an exemplary embodiment.

[0054] Figure 6 is a schematic diagram showing the interaction between a ligand and surrounding residues during a molecular dynamics simulation according to an exemplary embodiment.

[0055] Figure 7 It is a block diagram of an automated molecular dynamics simulation system for an enzyme reaction intermediate complex according to an exemplary embodiment.

[0056] Figure 8 It is a block diagram of an electronic device according to an exemplary embodiment. DETAILED DESCRIPTION

[0057] The specific implementation of the present disclosure is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0058] Figure 1 is a flow chart of an automated molecular dynamics simulation method for an enzyme reaction intermediate complex according to an exemplary embodiment. Figure 2 is another flow chart of an automated molecular dynamics simulation method for an enzyme reaction intermediate complex according to an exemplary embodiment, see Figure 1 and Figure 2 , the method comprising:

[0059] S101, obtaining an input protein ligand structure file containing a P450 enzyme, parsing the protein ligand structure file to obtain a heme structure file, a common ligand structure file, and a pure protein structure file of the P450 enzyme.

[0060] S102, preprocessing the pure protein structure file, the heme structure file and the common ligand structure file respectively.

[0061] S103, obtaining a mutation setting file, mutating the designated sites in the preprocessed purified protein structure file based on the mutation setting file, and performing a second preprocessing on the mutated purified protein structure file.

[0062] S104, based on the preset quantum mechanics QM calculation result file, modify the pure protein structure file after the second preprocessing and the preprocessed heme structure file to obtain the intermediate pure protein structure file and the intermediate heme structure file respectively, and assemble the molecular dynamics simulation input file containing the intermediate complex structure based on the intermediate pure protein structure file, the intermediate heme structure file and the preprocessed common ligand structure file.

[0063] S105, generating constraint files and index files for molecular dynamics simulation, constructing a molecular dynamics simulation process and generating an automated script.

[0064] S106, running the automated script to perform molecular dynamics simulation calculations on the input file to obtain a calculation result file.

[0065] S107, read the operation result file, calculate the target parameters and perform visual display.

[0066] In step S101, a protein ligand structure file in a PDB format input by a user may be received, wherein the protein ligand structure file includes structures of proteins, heme and other common ligands, wherein the protein is a P450 enzyme. The protein ligand structure file may be a protein ligand complex structure file including a P450 enzyme, or may be a set of files including a heme structure file, a common ligand structure file and a pure protein structure file of a P450 enzyme. By automatically identifying the components of the input structure file, these files may be parsed by splitting, extracting and the like, thereby obtaining independent pure protein structure files of P450 enzymes, heme structure files and common ligand structure files.

[0067] Optionally, in step S101, the protein ligand structure file is parsed to obtain a heme structure file, a common ligand structure file, and a pure protein structure file of a P450 enzyme, including:

[0068] If the protein-ligand structure file is a protein-ligand complex structure file, the P450 enzyme structure in the protein-ligand complex structure file is identified, and the pure protein structure of the P450 enzyme is retained according to the standard residues and the blocking residues to obtain the pure protein structure file. Based on the HETATM identifier, the heme structure and other ligand structures in the reaction pocket in the protein-ligand complex structure file are identified and extracted to obtain the heme structure file and the common ligand structure file, respectively.

[0069] Specifically, the protein-ligand complex structure file contains the complex structure, which needs to be automatically split. The protein-ligand complex structure file can be read and loaded, such as a PDB format file, and the structure of the P450 enzyme in the file can be identified based on standard residues and capping residues. The protein structure of the identified P450 enzyme is retained to obtain a pure protein structure file. Then the HETATM logo is identified, the heme structure in the reaction pocket is extracted, and retained to obtain a heme structure file, and other common ligand structures are identified and retained to obtain a common ligand structure file.

[0070] The protein-ligand structure file may be a file set composed of multiple independent structure files uploaded separately. At this time, the required structure files such as heme structure files, ligand structure files and pure protein structure files of P450 enzymes can be automatically identified to distinguish them from other structure files such as water molecule structure files.

[0071] After step S101 is completed, step S102 can be executed. In step S102, common ligands, heme ligands and pure protein structures can be preprocessed, and preparation files can be generated to ensure the quality of the input structure and make it more suitable for subsequent computational analysis. For example, all parsed ligand small molecules can be neutralized, reordered, and standardized using reduce or openbabel, and all possible topology files and mol2 structure files can be generated using acpype. Using the mol2 file of the ligand small molecule, write and call tleap to build the corresponding lib file.

[0072] Optionally, in step S102, the pure protein structure file, the heme structure file and the common ligand structure file are preprocessed respectively, including:

[0073] S1021, protonating the pure protein structure of the pure protein structure file, and reordering the residues of the pure protein structure with disrupted sequence or discontinuity, to obtain a pre-processed pure protein structure file.

[0074] S1022, hydrogenating the heme structure file and adjusting the atom numbers to obtain a preprocessed heme structure file.

[0075] S1023, neutralize the common ligand structure file to obtain the preprocessed common ligand structure file, generate the structure file and topology file of the ligand molecule based on the preprocessed common ligand structure file, generate the corresponding tleap configuration file based on the structure file, and run the tleap configuration file to obtain the corresponding lib file.

[0076] In step S1021, the protein structure can first be protonated using pdbfixer. If the residue order is disrupted or there is discontinuity, pdb4amber is used to reorder the residues to obtain a pre-processed pure protein structure. After step S1021 is completed, step S1022 can be executed.

[0077] In step S1022, reduce can be used to hydrogenate the heme structure file and adjust the atom number to obtain the pre-processed heme structure file. After step S1022 is completed, step S1023 can be executed.

[0078] In step S1023, reduce can be used to neutralize the ligand molecules in the common ligand structure file, specifically hydrogenation can be performed to obtain the pretreated common ligand structure file. Then the pretreated common ligand structure file can be used as input, and acpype can be used to prepare all possible structure files and topology files for the ligand molecules for subsequent molecular dynamics simulation operations. Use the mol2 file in its structure file to write a tleap configuration file, and run tleap to generate the corresponding lib file.

[0079] After step S102 is completed, step S103 can be executed. In step S103, the mutation setting file input by the user can be obtained and parsed, and the mutation site and mutation direction can be obtained from it according to the fixed rule text, and then pymol can be used to perform residue mutation on the specified site of the pre-processed pure protein structure file. The mutation setting file can contain information such as the specified site. After the mutation, the pure protein structure file after the mutation can be pre-processed for the second time with reference to step S1021.

[0080] After step S103 is completed, step 104 can be executed. In step S104, a parameterized state preparation file is prepared according to the intermediate state selected by the user. The quantum mechanics QM (Quantum Mechanical) calculation result file is calculated based on the principle of quantum mechanics and contains the actual situation of the CPDI (Compound I) state. Optionally, in step S104, based on the preset quantum mechanics QM calculation result file, the pure protein structure file after the second pretreatment and the pretreated hemoglobin structure file are modified to obtain the intermediate pure protein structure file and the intermediate hemoglobin structure file, respectively, including:

[0081] S1041, based on the preset quantum mechanics QM calculation result file, the hydrogen atom named HG in the 400th CYS residue in the pure protein structure file after the second preprocessing is deleted to match the actual situation of the CPDI state of the QM calculation result file, and the CYS residue with the hydrogen atom deleted is renamed CYM to obtain the intermediate pure protein structure file.

[0082] S1042, based on the preset quantum mechanics QM calculation result file, match the atomic names in the preprocessed heme structure file, modify the atomic order in the preprocessed heme structure file according to the atomic order in the QM calculation result file, add the O1 atom in the preprocessed heme structure file corresponding to the CPDI state of the QM calculation result file, and establish a connection relationship between the O1 atom and the divalent high-spin iron atom in the preprocessed heme structure file to obtain the intermediate state heme structure file.

[0083] In step S1041, the intermediate residues can be corrected. If the protein structure of a specific intermediate state is different from the crystal structure, the hydrogen atoms that do not exist in the intermediate state are deleted from the CYS residues to prepare a matching state file, i.e., a QM calculation result file. For details, see Figure 3A , the hydrogen atom named HG in the 400th CYS residue is deleted to match the actual situation of the CPDI state, and the residue is renamed CYM to obtain the intermediate pure protein structure file. After step S1041 is completed, step S1042 can be executed.

[0084] In step S1042, see Figure 3B , the original heme structure file matches the atom names according to the preset QM calculation result file, and modifies the atomic order in the heme structure file according to the atomic order. Add the O1 atom in the CPDI state and establish a connection relationship with the iron atom in the divalent high-spin state to obtain the intermediate heme structure file. For example, if the PDB file cannot be directly bonded, the iron atom and the oxygen atom can be connected in the frcmod file in the form of atomic type connection after adding the O1 atom, and then the connection relationship between the two can be established.

[0085] In step S104, after the intermediate pure protein structure file and the intermediate heme structure file are prepared, the intermediate pure protein structure file, the intermediate heme structure and the pre-processed common ligand structure file are assembled, and the relationship between these components is constructed to obtain the complex structure, and the parameters such as environmental variables are constructed by relevant software to form the final input file for molecular dynamics simulation. Optionally, in step S104, the molecular dynamics simulation input file containing the intermediate complex structure is assembled based on the intermediate pure protein structure file, the intermediate heme structure file and the pre-processed common ligand structure file, including:

[0086] S104a, assembling the intermediate pure protein structure file, the intermediate heme structure file and the pre-treated common ligand structure file to obtain the intermediate complex structure file.

[0087] S104b, construct the tleap input file of the complex of pure protein, hemoglobin and ligand, obtain the force field of the protein, small molecule and water selected by the user and load it using the source parameter, use loadamberparams to load the intermediate complex structure file, lib file, structure file and topology file of the ligand molecule, use loadpdb to load the preset complex template structure, use addions to automatically add the neutralized ion model of the force field, use slovatebox to load the specified water box model, and finally use saveamberparm to generate the coordinate file and topology file in Amber format as the molecular dynamics simulation input file containing the intermediate complex structure.

[0088] In step S104a, the intermediate pure protein structure file, the intermediate heme structure file and the pre-processed common ligand structure file can be put into the same text to complete the assembly, and then the atom numbering can be readjusted. After step S104a is completed, step S104b can be executed.

[0089] In step S104b, a tleap input file can be prepared according to the previous preparation, and the position parameters of the protein, ligand, and water can be specified according to the user's choice and loaded using the source parameter. Use loadamberparams to load the assembled intermediate complex structure file, lib file, ligand molecule structure file and topology file such as fracmod file, mol2 and lib files of other ligands, etc., import the intermediate CYM residue and heme structure file, load the complex template structure, neutralized ion model and water box model, and the water box model can choose regular octahedron, regular dodecahedron and other models. Finally, use saveamberparm to generate the coordinate file and topology file in Amber format as the molecular dynamics simulation input file. This file not only contains the assembled complex, but also contains environmental parameters, etc. It can be directly used as the input file for subsequent molecular dynamics simulation operations. The complex system can be found in Figure 4 .

[0090] After step S104 is completed, step S105 can be executed. In step S105, the preparation of relevant preparation files and the construction of molecular dynamics simulation process are mainly carried out, and an automated script is generated. Specifically, in step S105, genrestr can be used to select protein group, ligand group, and C_alpha group respectively, generate corresponding itp format constraint files, and use makendx to prepare ndx format index files for protein+heme+ligand group.

[0091] Then, you can build a preset molecular dynamics simulation process, for example, it can include energy minimization steps, ensemble equilibrium steps and production simulation steps. The energy minimization step can include energy minimization steps for pure protein + ligand, energy minimization steps for low thresholds and energy minimization steps for high thresholds. The ensemble equilibrium step can include NVE ensemble equilibrium step, NVT ensemble equilibrium step and NPT ensemble equilibrium step. After setting the steps, the corresponding energy minimization, ensemble equilibrium and multi-step simulation mdp files can be generated according to the parameters modified by the user. Based on the above content, such as constraint files, index files and files of molecular dynamics simulation process, a run control bash automation script is generated.

[0092] After step S105 is completed, step S106 can be executed. In step S106, automated simulation can be performed in the production environment. The preset simulation environment and parameter settings in the environment are called, and the automated script is run to start the automated molecular dynamics simulation. During the process, the molecular dynamics simulation input file containing the intermediate state complex structure is automatically calculated, and the aforementioned prepared file is used. After the final calculation is completed, the calculation result file, such as a trajectory file in xtc format, can be obtained.

[0093] After step S106 is completed, step S107 may be executed. In step S107, the required target parameters may be calculated and visualized based on the operation result file such as the trajectory file. For example, a python script may be called to visualize the regioselectivity, distance calculation, residue interaction, etc. Optionally, in step S107, the operation result file is read, the target parameters are calculated, and the visualization is performed, including:

[0094] S1071, read the calculation result file, use RDKit to label the atomic number of the ligand of the regional selectivity result, and use PyMol to complete the visualization.

[0095] S1072, extracting the atomic coordinates in the calculation result file, calculating the distance between the ligand atom and the intermediate heme O1 atom based on the atomic coordinates, and visualizing it using matplotlib.

[0096] S1073, calculate the minimum distance pair between the ligand atom and the surrounding residues in the operation results, retain the records of interactions during the simulation time according to the distance threshold modified by the user, and use matplotlib to visualize the interaction between the ligand and the surrounding residues.

[0097] In step S1071, see Figure 5A , you can use pymol to visualize the ligands of the regioselectivity results, and use rdkit to add atomic numbers.

[0098] In step S1072, see Figure 5B , using the previous example, we can extract the atomic coordinates in the trajectory file, calculate the distance between the ligand atom and the intermediate heme O1 atom, and use matplotlib for visualization.

[0099] In step S1073, see Figure 6 , the minimum distance pair between the ligand atom and the surrounding residues can be calculated, and the records of interactions in the simulation time can be retained according to the distance threshold modified by the user, and the interactions between the ligand and the surrounding residues can be visualized using matplotlib. Among them, retaining the records of interactions in the simulation time according to the distance threshold modified by the user can include: retaining the records of interactions in the simulation time that is less than or equal to the distance threshold modified by the user.

[0100] Through the above technical solution, after the pure protein structure file, heme structure file and common ligand structure file of P450 enzyme are parsed, these structure files are preprocessed, mutated and modified in the intermediate state respectively, and finally the molecular dynamics simulation input file containing the intermediate state complex structure is assembled based on the intermediate state pure protein structure file, the intermediate state heme structure file and the preprocessed common ligand structure file, and then the molecular dynamics simulation operation script is prepared, and the input file is subjected to molecular dynamics simulation calculation by running the script to obtain the operation result file, and the target parameters can be calculated and visualized based on the operation result file, so as to construct a set of ligand reaction selectivity prediction process and intermediate state complex system simulation method suitable for P450 enzyme complex system, and the MD simulation of intermediate state complex is completed by the automation, so that the experimenter can also perform professional calculation simulation prediction, and reduce the experimental cost. The technical solution also provides the visualization results of the interaction, is not limited to the common bond length and bond angle calculation, and simplifies the difficulty of interpreting the steric constraint.

[0101] Figure 7 is a block diagram of an automated molecular dynamics simulation system for an enzyme reaction intermediate complex according to an exemplary embodiment, see Figure 7 The system comprises:

[0102] A structure file parsing module, used for obtaining an input protein ligand structure file containing a P450 enzyme, parsing the protein ligand structure file to obtain a heme structure file, a common ligand structure file, and a pure protein structure file of the P450 enzyme;

[0103] The structure preprocessing module is used to preprocess the pure protein structure file, heme structure file and common ligand structure file respectively;

[0104] A protein mutation module, used to obtain a mutation setting file, mutate a specified site in the preprocessed pure protein structure file based on the mutation setting file, and perform a second preprocessing on the mutated pure protein structure file;

[0105] The intermediate state preparation module is used to modify the pure protein structure file after the second pretreatment and the heme structure file after the pretreatment based on the preset QM calculation result file, and obtain the intermediate state pure protein structure file and the intermediate state heme structure file respectively, and assemble the molecular dynamics simulation input file containing the intermediate state complex structure based on the intermediate state pure protein structure file, the intermediate state heme structure file and the pretreated common ligand structure file;

[0106] Simulation building module, used to generate constraint files and index files for molecular dynamics simulation, build molecular dynamics simulation process and generate automation scripts;

[0107] An automated molecular dynamics simulation module, used for running the automated script to perform a molecular dynamics simulation operation on the input file to obtain an operation result file;

[0108] The predefined result analysis module is used to read the operation result file, calculate the target parameters and display them visually.

[0109] Optionally, the protein-ligand structure file is a protein-ligand complex structure file containing a P450 enzyme or a set of files containing a heme structure file, a common ligand structure file and a pure protein structure file of a P450 enzyme, and the structure file parsing module is further used for:

[0110] If the protein-ligand structure file is a protein-ligand complex structure file, the P450 enzyme structure in the protein-ligand complex structure file is identified, and the pure protein structure of the P450 enzyme is retained according to the standard residues and the blocking residues to obtain the pure protein structure file. Based on the HETATM identifier, the heme structure and other ligand structures in the reaction pocket in the protein-ligand complex structure file are identified and extracted to obtain the heme structure file and the common ligand structure file, respectively.

[0111] Optionally, the structure preprocessing module is further used for:

[0112] Protonate the pure protein structure of the pure protein structure file, and reorder the residues of the pure protein structure with disrupted sequence or discontinuity to obtain a pre-processed pure protein structure file;

[0113] Hydrogenate the heme structure file and adjust the atom number to obtain the preprocessed heme structure file;

[0114] The common ligand structure file is neutralized to obtain the preprocessed common ligand structure file, and the structure file and topology file of the ligand molecule are generated based on the preprocessed common ligand structure file, and the corresponding tleap configuration file is generated based on the structure file, and the tleap configuration file is run to obtain the corresponding lib file.

[0115] Optionally, the intermediate state preparation module is further used for:

[0116] Based on the preset quantum mechanics QM calculation result file, the hydrogen atom named HG in the 400th CYS residue in the pure protein structure file after the second preprocessing is deleted to match the actual situation of the CPDI state of the QM calculation result file, and the CYS residue with the hydrogen atom deleted is renamed CYM to obtain an intermediate pure protein structure file;

[0117] Based on the preset QM calculation result file, the atomic names in the preprocessed heme structure file are matched, the atomic order in the preprocessed heme structure file is modified according to the atomic order in the QM calculation result file, the O1 atom corresponding to the CPDI state of the QM calculation result file is added to the preprocessed heme structure file, and a connection relationship is established between the O1 atom and the divalent high-spin iron atom in the preprocessed heme structure file to obtain an intermediate heme structure file.

[0118] Optionally, the intermediate state preparation module is further used for:

[0119] Assembling the intermediate pure protein structure file, the intermediate heme structure file and the pre-treated common ligand structure file to obtain the intermediate complex structure file;

[0120] Build the tleap input file of the complex of pure protein, hemoglobin and ligand, obtain the force field of protein, small molecule and water selected by the user and load it using source parameters, use loadamberparams to load the intermediate complex structure file, lib file, structure file and topology file of ligand molecule, use loadpdb to load the preset complex template structure, use addions to automatically add the neutralized ion model of the force field, use slovatebox to load the specified water box model, and finally use saveamberparm to generate the coordinate file and topology file in Amber format as the molecular dynamics simulation input file containing the intermediate complex structure.

[0121] Optionally, the molecular dynamics simulation process includes an energy minimization step, an ensemble balancing step and a production simulation step which are performed sequentially.

[0122] Optionally, the ensemble equilibration step includes an NVE ensemble equilibration step, an NVT ensemble equilibration step and an NPT ensemble equilibration step.

[0123] Optionally, the predefined result analysis module is further used to:

[0124] Read the calculation result file, use RDKit to label the atomic number of the ligand of the regional selectivity result, and use PyMol to complete the visualization;

[0125] Extract the atomic coordinates in the calculation result file, calculate the distance between the ligand atom and the intermediate heme O1 atom based on the atomic coordinates, and use matplotlib for visualization;

[0126] The minimum distance pair between the ligand atom and the surrounding residues in the calculation results is calculated. The records of interactions during the simulation time are retained according to the distance threshold modified by the user. The interaction between the ligand and the surrounding residues is visualized using matplotlib.

[0127] Regarding the device in the above embodiment, the specific manner in which each module performs operations has been described in detail in the embodiment of the method, and will not be elaborated here.

[0128] Figure 8 FIG. 1 is a block diagram of an electronic device according to an exemplary embodiment. Figure 8 As shown, the electronic device 700 may include: a processor 701 , a memory 702 . The electronic device 700 may also include one or more of a multimedia component 703 , an input / output (I / O) interface 704 , and a communication component 705 .

[0129] The processor 701 is used to control the overall operation of the electronic device 700 to complete all or part of the steps in the above-mentioned enzyme reaction intermediate complex automated molecular dynamics simulation method. The memory 702 is used to store various types of data to support the operation of the electronic device 700, and these data may include instructions for any application or method used to operate on the electronic device 700, as well as application-related data, such as contact data, sent and received messages, pictures, audio, video, etc. The memory 702 can be implemented by any type of volatile or non-volatile storage device or a combination thereof, such as static random access memory (Static Random Access Memory, referred to as SRAM), electrically erasable programmable read-only memory (Electrically Erasable Programmable Read-Only Memory, referred to as EEPROM), erasable programmable read-only memory (Erasable Programmable Read-Only Memory, referred to as EPROM), programmable read-only memory (Programmable Read-Only Memory, referred to as PROM), read-only memory (Read-Only Memory, referred to as ROM), magnetic memory, flash memory, disk or optical disk. The multimedia component 703 may include a screen and an audio component. The screen may be, for example, a touch screen, and the audio component is used to output and / or input audio signals. For example, the audio component may include a microphone, which is used to receive external audio signals. The received audio signal may be further stored in the memory 702 or sent through the communication component 705. The audio component also includes at least one speaker for outputting audio signals. The I / O interface 704 provides an interface between the processor 701 and other interface modules, and the above-mentioned other interface modules may be keyboards, mice, buttons, etc. These buttons may be virtual buttons or physical buttons. The communication component 705 is used for wired or wireless communication between the electronic device 700 and other devices. Wireless communication, such as Wi-Fi, Bluetooth, Near Field Communication (NFC), 2G, 3G, 4G, NB-IOT, eMTC, or other 5G, etc., or a combination of one or more of them, is not limited here. Therefore, the corresponding communication component 705 may include: Wi-Fi module, Bluetooth module, NFC module, etc.

[0130] In an exemplary embodiment, the electronic device 700 can be implemented by one or more application specific integrated circuits (ASIC), digital signal processors (DSP), digital signal processing devices (DSPD), programmable logic devices (PLD), field programmable gate arrays (FPGA), controllers, microcontrollers, microprocessors or other electronic components to execute the above-mentioned automated molecular dynamics simulation method of enzyme reaction intermediate complexes.

[0131] In another exemplary embodiment, a computer-readable storage medium including program instructions is also provided, and when the program instructions are executed by a processor, the steps of the above-mentioned method for automated molecular dynamics simulation of enzyme reaction intermediate complexes are implemented. For example, the computer-readable storage medium can be the above-mentioned memory 702 including program instructions, and the above-mentioned program instructions can be executed by the processor 701 of the electronic device 700 to complete the above-mentioned method for automated molecular dynamics simulation of enzyme reaction intermediate complexes.

[0132] The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0133] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0134] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A method for automated molecular dynamics simulation of enzyme reaction intermediate complexes, characterized in that: The method comprises: Obtaining an input protein ligand structure file containing a P450 enzyme, parsing the protein ligand structure file to obtain a heme structure file, a common ligand structure file, and a pure protein structure file of the P450 enzyme; Preprocess the pure protein structure file, heme structure file and common ligand structure file respectively; Obtaining a mutation setting file, mutating a designated site in the preprocessed purified protein structure file based on the mutation setting file, and performing a second preprocessing on the mutated purified protein structure file; Based on the preset quantum mechanics QM calculation result file, the hydrogen atom named HG in the 400th CYS residue in the pure protein structure file after the second preprocessing is deleted to match the actual situation of the CPDI state of the QM calculation result file, and the CYS residue with the hydrogen atom deleted is renamed CYM to obtain an intermediate pure protein structure file; Based on the preset QM calculation result file, the atomic names in the preprocessed heme structure file are matched, the atomic order in the preprocessed heme structure file is modified according to the atomic order in the QM calculation result file, the O1 atom corresponding to the CPDI state of the QM calculation result file is added to the preprocessed heme structure file, and the O1 atom is connected to the divalent high-spin iron atom in the preprocessed heme structure file to obtain an intermediate state heme structure file; Based on the intermediate pure protein structure file, the intermediate heme structure file and the pre-processed common ligand structure file, a molecular dynamics simulation input file containing the intermediate complex structure is assembled; Generate constraint files and index files for molecular dynamics simulation, build molecular dynamics simulation process and generate automation scripts; Running the automated script to perform a molecular dynamics simulation operation on the input file to obtain an operation result file; Read the operation result file, calculate the target parameters and display them visually.

2. The method according to claim 1, characterized in that The protein ligand structure file is a protein ligand complex structure file containing a P450 enzyme or a set of files containing a heme structure file, a common ligand structure file and a pure protein structure file of a P450 enzyme. The parsing of the protein ligand structure file to obtain the heme structure file, the common ligand structure file and the pure protein structure file of the P450 enzyme comprises: If the protein-ligand structure file is a protein-ligand complex structure file, the P450 enzyme structure in the protein-ligand complex structure file is identified, and the pure protein structure of the P450 enzyme is retained according to the standard residues and the blocking residues to obtain the pure protein structure file. Based on the HETATM identifier, the heme structure and other ligand structures in the reaction pocket in the protein-ligand complex structure file are identified and extracted to obtain the heme structure file and the common ligand structure file, respectively.

3. The method according to claim 1, characterized in that The preprocessing of the pure protein structure file, the heme structure file and the common ligand structure file respectively includes: Protonate the pure protein structure of the pure protein structure file, and reorder the residues of the pure protein structure with disrupted sequence or discontinuity to obtain a pre-processed pure protein structure file; Hydrogenate the heme structure file and adjust the atom number to obtain the preprocessed heme structure file; The common ligand structure file is neutralized to obtain the preprocessed common ligand structure file, and the structure file and topology file of the ligand molecule are generated based on the preprocessed common ligand structure file, and the corresponding tleap configuration file is generated based on the structure file, and the tleap configuration file is run to obtain the corresponding lib file.

4. The method according to claim 3, characterized in that The molecular dynamics simulation input file containing the intermediate state complex structure is assembled based on the intermediate state pure protein structure file, the intermediate state heme structure file and the pre-processed common ligand structure file, including: Assembling the intermediate pure protein structure file, the intermediate heme structure file and the pre-treated common ligand structure file to obtain the intermediate complex structure file; Build the tleap input file of the complex of pure protein, hemoglobin and ligand, obtain the force field of protein, small molecule and water selected by the user and load it using source parameters, use loadamberparams to load the intermediate complex structure file, lib file, structure file and topology file of ligand molecule, use loadpdb to load the preset complex template structure, use addions to automatically add the neutralized ion model of the force field, use slovatebox to load the specified water box model, and finally use saveamberparm to generate the coordinate file and topology file in Amber format as the molecular dynamics simulation input file containing the intermediate complex structure.

5. The method according to claim 1, characterized in that The molecular dynamics simulation process includes an energy minimization step, an ensemble balancing step and a production simulation step which are performed sequentially.

6. The method according to claim 5, characterized in that The ensemble equilibrium step includes an NVE ensemble equilibrium step, an NVT ensemble equilibrium step and an NPT ensemble equilibrium step.

7. The method according to claim 1, characterized in that The reading of the operation result file, calculation of the target parameters and visual display thereof include: Read the calculation result file, use RDKit to label the atomic number of the ligand of the regional selectivity result, and use PyMol to complete the visualization; Extract the atomic coordinates in the calculation result file, calculate the distance between the ligand atom and the intermediate heme O1 atom based on the atomic coordinates, and use matplotlib for visualization; The minimum distance pair between the ligand atom and the surrounding residues in the calculation results is calculated. The records of interactions during the simulation time are retained according to the distance threshold modified by the user. The interaction between the ligand and the surrounding residues is visualized using matplotlib.

8. An automated molecular dynamics simulation system for enzyme reaction intermediate complexes, characterized in that: The system comprises: A structure file parsing module, used for obtaining an input protein ligand structure file containing a P450 enzyme, parsing the protein ligand structure file to obtain a heme structure file, a common ligand structure file, and a pure protein structure file of the P450 enzyme; The structure preprocessing module is used to preprocess the pure protein structure file, heme structure file and common ligand structure file respectively; A protein mutation module, used for obtaining a mutation setting file, mutating a designated site in the preprocessed pure protein structure file based on the mutation setting file, and performing a second preprocessing on the mutated pure protein structure file; The intermediate state preparation module is used to delete the hydrogen atom named HG in the 400th CYS residue in the pure protein structure file after the second pretreatment based on the preset quantum mechanics QM calculation result file to match the actual situation of the CPDI state of the QM calculation result file, and rename the CYS residue with the hydrogen atom deleted to CYM to obtain the intermediate state pure protein structure file; based on the preset QM calculation result file, match the atom name in the pretreated heme structure file, modify the atom order in the pretreated heme structure file according to the atom order in the QM calculation result file, add the O1 atom in the pretreated heme structure file corresponding to the CPDI state of the QM calculation result file, and establish a connection relationship between the O1 atom and the divalent high-spin iron atom in the pretreated heme structure file to obtain the intermediate state heme structure file; assemble the molecular dynamics simulation input file containing the intermediate state complex structure based on the intermediate state pure protein structure file, the intermediate state heme structure file and the pretreated common ligand structure file; Simulation building module, used to generate constraint files and index files for molecular dynamics simulation, build molecular dynamics simulation process and generate automation scripts; An automated molecular dynamics simulation module, used for running the automated script to perform a molecular dynamics simulation operation on the input file to obtain an operation result file; The predefined result analysis module is used to read the operation result file, calculate the target parameters and display them visually.

9. An electronic device, characterized in that: include: a memory having a computer program stored thereon; A processor, configured to execute the computer program in the memory to implement the steps of the method according to any one of claims 1 to 7.

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