METHOD FOR OPTIMIZING THE CONDITIONS OF PROTEIN CRYSTALLIZATION USING THE MOLECULAR DYNAMICS METHOD Russian patent published in 2022 - IPC G16B5/30 C30B7/00 C30B29/58 

FIELD: biochemistry.

SUBSTANCE: invention relates to methods for optimizing the conditions of protein crystallization for the subsequent growth of a protein crystal under optimized conditions and decoding the protein structure with high resolution using X-ray diffraction analysis and can be applied in the field of protein crystallography. To achieve it, a method has been proposed to optimize the conditions of protein crystallization using the molecular dynamics method, which consists in isolating oligomers contained in a pre-crystallization solution, calculating molecular dynamics and changes in free energy during the formation of an oligomer under various pH conditions, precipitator type, precipitator concentration, temperature according to the following protocol, which consists in the fact that for each simulated crystallization conditions, the pH of the system is first set by determining the states of protonation of amino acid residues in oligomers using the PROPKA program, after which all calculations and preparation of structures for them are performed in the GROMACS software package, the prepared structures are placed in the center of the modeling cell, with the minimum distance between its edge and the protein molecule being 1 nm, at the same time, the rest of the cell space is filled with water, precipitator components are added to the system in the required concentration, energy and NVT and NPT-system balancing are minimized, the productive MD is calculated in the NPT ensemble, while integration is performed according to the standard leap-frog algorithm with a time step of integration of 2 fs and the duration of the calculated trajectories of 100 ns, while before analyzing the obtained trajectories, artifacts arising from quasi-infinite periodic boundary conditions are eliminated, according to the data of the obtained trajectories graphs of the root-mean-square fluctuations of C_α atoms (RMSF) are plotted and energy changes are calculated during the formation of protein oligomers, at the same time, the MM/GBSA method is used to calculate the change in free energy during the formation of oligomers, the free energy is calculated for each trajectory according to the results of MD modeling, using the GROMACS program, on 1000 frames using the gmx_MMPBSA module version 1.4.0 in combination with the MMPBSA.py script and the AmberTools20 package, optimal parameters for the crystallization of the protein under study are established, at which the smallest root-mean-square fluctuations of oligomer atoms and the largest decrease in free energy are observed.

EFFECT: reduction in the cost and acceleration of the stage of optimization screening of protein crystallization conditions.

1 cl, 2 dwg, 3 tbl, 1 ex