Before the molecular dynamics (MD) simulation, PRMT5 was centered and solvated in cubic box using TIP3P water. Then, energy minimization was performed in Gromacs 4.5 (61) using the all-atom Amber99sb FF (62) with steepest descent algorithm, which allows fast convergence to the local energy minima. Next, the equilibration of the system was performed in two steps: 100-ps canonical ensemble (NVT) and 100-ps isothermal-isobaric ensemble (NPT) simulation with harmonic restraints (force constant, 1 kJ mol−1 K−1) applied to the backbone atoms of the protein. Furthermore, the production MD runs were carried out for 100 ns with the NPT ensemble without any restraints.

During all simulations, a 2-fs integration time step was used. The temperature was maintained at 300 K with the V-rescale algorithm, and the pressure was coupled at 1 bar by isotropic pressure coupling using the Parrinello-Rahman algorithm (time constant, 10 ps; isothermal compressibility of water, 4.5 × 10−5 bar−1). Long-range electrostatics was calculated by fourth-order particle mesh Ewald algorithm with a grid spacing of 0.16 nm. All bonds were constrained using a fourth-order P-LINCS algorithm. Electrostatic and van der Waals interactions were cut off at 1 nm, and a dispersion correction was applied to account for it. Periodic boundary conditions were implemented in three dimensions. Initial atomic velocities, before NVT equilibration, were obtained from Maxwell’s distribution at 310 K. Trajectories were analyzed by Gromacs tools, Pymol, VMD, and custom Bash shell and Python (python.org) scripts using NumPy (numpy.org) and Matplotlib libraries.

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