Er two docking applications didn’t incorporate energy minimization procedures. The PatchDock’ model was one of the most perturbed, as when compared with the outcome on the docking routine, due to the manual editing, which may well explain the pronounced impact of energy minimization. 24) I don’t assume 45 ns is really a extended enough simulation to say something about stability of your entire complicated, specifically provided the massive size of this complex. 25) “.. As a result, MD simulations revealed only one model (the PatchDock’ model, Fig. 1) that kept the proper domain architecture and intact geometry during the MD simulation..” this worries me. Could it be that a far more cautious equilibration of MD is necessary Or that the complexes are wrong Authors’ response: As we have explicitly emphasized in the revised manuscript, the model structures could be all wrong, they may be just theoretical predictions that await experimental scrutiny. Our job was, having said that, to identify the residues of Apaf-1 which are involved in binding of cytochrome c. We think that we’ve solved this trouble by combining structural modeling with sequence evaluation. We had to limit our MD simulation time for you to 45 ns as a N-(2-Hydroxypropyl)methacrylamide Biological Activity result of significant size on the program. Nonetheless, we believe thatthe simulation time was enough to discriminate a mechanically “wrong” structure from a stable one particular. The heat maps in More file 1: Figure S1 show that though the stability with the ClusPro structure decreased with time, the stability from the PatchDock’ structure enhanced via the MD simulation. So it seems unlikely that the PatchDoc’ structure would break up upon a longer MD simulation. 26) “..of Apaf-1 is extra or significantly less evenly negatively charged..” much more or significantly less Deleted 27) “..correlation coefficient of 0.9463 as when compared with 0.9558..” how calculated Authors’ response: We have utilized UCSF Chimera package [84]. The reference to this application has been added for the Procedures section. 28) Error: “.. Electrostaticpolar interactions or bonds that contain salt 4-Ethyloctanoic acid Autophagy bridges and prospective H-bonds are typically regarded inside a four cutoff..” the 4A cutoff is for H-bonds. Salt bridges have a tendency to possess a cutoff of 8-12A and even longer. The shorter salt bridges occasionally are named H-bonded salt bridges. This also why there need to be at the least 12A between the solute and the simulation box… Authors’ response: We don’t see an error right here. The criterion for identifying a salt bridge, as originally proposed by Barlow and Thornton [54], is the fact that the distance among the heavy atoms with the ionizable groups of charged residues really should be less than 4 This cut-off of 4 has been utilised for defining salt bridges in a lot of research, see [503] and references therein, also as within the preceding studies of cytochrome c interactions with its partners [42]. The cut-off of four was also taken for salt bridges within the paper of de Groot and co-workers [49] that was co-authored by the Reviewer. We’ve added the references to all these classical papers to the revised manuscript. It’s essential to note that we also discuss the long-range interactions. In the original manuscript, we’ve got thought of a cut-off of five as experimental research show detectable interactions even at this distance [55], also for the 3 cut-off utilized to identify strong hydrogen bonds (Table three inside the revised manuscript). To address this comment with the Reviewer, within the revised manuscript, we have added the data that had been collected having a cut-off of 6 to illustrate that any further improve within the cut-offShalae.