3.7.1 MD simulation
The MD simulation was performed using GROMACS 5.1.4 on in-house super computing platform as earlier implementing the GROMACS 53a6 force field.58 The initial structure was solvated with a simple point-charge model of SPC water in a box. Na+ions were added to neutralize the negative charges in the system. The system was then subjected to a steepest descent energy minimization to give the maximum force below 1000 KJ/mol/nm. After energy minimization, the position restraint simulation of 2000 steps (2fs each steep) was implemented under NVT (the constant Number of particles, Volume and Temperature) and NPT (the constant Number of particles, Pressure and Temperature) condition. In the end, 50ns MD simulation was conducted on each ensemble. The root means square deviation (RMSD) and the root mean square fluctuation (RMSF), which is an important index for evaluating the protein structure, were calculated using GROMACS rms and rmsf tools. Time dependent secondary structure was calculated using do_dssp tool. The Particle Mesh Ewald method was used calculated long-range electrostatic forces. The neighbor list was determined with the Grid method. The time step of the simulations was 2 fs, and the coordinates were saved for analysis every 2 ps. Postprocessing and analysis were performed using standard GROMACS tools and Visual Molecular Dynamics (VMD).