Dear Sir/Madam,

No Doubt, I m new in Groamcs and a new learner. A manuscript of mine is under major revision. The reviewer has raised some query in the Material & Method Section. The Methods Section and Reviewers Comment are below. Please help me in rectifying the mistake and the changes required. The PDB ID of the protein is 2AZ5 (TNF-alpha).

========================Method=========================

All MD simulations were carried out on Ubuntu. MD simulation for the protein and the protein-ligand complex was carried out using GROMACS version 5.1.2. Initially, the Protein Preparation Tool in the MVD 6.0.1 was used to stabilize and energy minimized both the protein and the protein-ligand docked complexes. The distorted residues viz. Asp10, Tyr87, Glu146 and Leu157 were automatically checked for their protonated states which were further repaired and rebuild using Protein Preparation Tool. For the individual enzyme, (PDB ID: 2AZ5), the topology file was processed with OPLS-AA/L all-atom force field (2001 amino acid dihedrals) and for the docked complex, it was processed with GROMOS96 43a1 force field.35,36. For the protein-ligand complex, initially, the coordinates of the ligands were separated and the topology of the ligand was generated using the PRODRG 2.5 server37. All the MD simulations were carried out using periodic boundary conditions. Solvation was accomplished by configuring using the Simple Point Charge water38 (SPC) solvent model in water molecules in a box with 64 x 64 x 64 Å3, counter-ionized in Na+ and Cl- ions at a concentration of 0.15 mol/L NaCl and equilibrated. Further, the solvated structures were energy minimized using the steepest descent minimization, terminating when the maximum force is < 10.0 kJ/mol. Next, the system was equilibrated with NPT ensemble phase at constant temperature (300 K) and pressure (1 bar) with a time step of 2 fs. Further, the systems were progressed with NVT simulation for 1 ns (nanoseconds), and the minimized structure was equilibrated with a timescale of 20 ns (nanoseconds). Throughout the simulations, all bonds were constrained using the LINCS constraint algorithm with an iteration value of 1 and order value of 4. The integration time step was set to 2 fs. The Particle Mesh Ewald (PME) for long-range electrostatic interactions was treated, with a cut-off value of 12 Å. The molecular dynamics simulation was performed on both the protein and the protein–ligand binding complex for 20 ns to understand the dynamic behavior of the protein and their stability. The trajectory were analyzed for their root mean square deviation (RMSD), root mean square fluctuations (RMSF), radius of gyration (Rg) and solvent accessible surface area (SASA).

=====================Reviewer's Query=====================

1) Regarding the stability of the monomeric specie, the authors mention that: “Yes, the stability of the monomeric structure was confirmed. It was minimized using a simple force field known as the PLP-potentials for steric and hydrogen bonding interactions, and the Coulomb potential for the electrostatic forces.” but this is not enough. What I was suggesting is to confirm if whether this specie should be stable of not. In other words, if the monomeric form is experimentally observed.

2) It is now written in the methods section that “The distorted residues viz. Asp10, Tyr87, Glu146 and Leu157 were automatically checked for their protonated states which were further repaired and rebuild using Protein Preparation Tool.”. I guess this means that Asp10, Glu146 and Cter (Leu157) were protonated during MD simulations which does not make any sense a priori. How were the protonation states of all residues assigned? In particular, how did the authors choose the protonation state of Histidine residues?

3) When the authors say “the minimized structure was equilibrated with a timescale of 20 ns” they mean that the total production time is 20 ns? This is not clear.

4) What was the Fourier spacing in PME?

5) “The Particle Mesh Ewald (PME) for long-range electrostatic interactions was treated, with a cut-off value of 12 Å.” This phrase is difficult to understand. I guess something like “The particle mesh Ewald (PME) was used, to treat long-range electrostatic interactions, with a cut-off value of 12 Å.” Also, how were the van der Walls interactions treated? With a plain cut-off at 12A?