Er two docking programs didn’t incorporate energy minimization procedures. The PatchDock’ model was essentially the most perturbed, as in comparison to the outcome of your docking routine, because of the manual editing, which might clarify the pronounced impact of energy minimization. 24) I never consider 45 ns is actually a extended adequate simulation to say something about stability on the whole complex, in particular given the enormous size of this complicated. 25) “.. Thus, MD simulations revealed only one particular model (the PatchDock’ model, Fig. 1) that kept the proper domain architecture and intact geometry throughout the MD simulation..” this worries me. Could it be that a considerably more cautious equilibration of MD is required Or that the complexes are wrong Authors’ response: As we’ve got explicitly emphasized inside the revised manuscript, the model structures might be all incorrect, they are just theoretical predictions that await experimental scrutiny. Our task was, however, to identify the residues of Apaf-1 which might be involved in binding of cytochrome c. We think that we have solved this problem by combining structural modeling with sequence analysis. We had to limit our MD simulation time for you to 45 ns because of the significant size from the technique. Nevertheless, we believe thatthe simulation time was adequate to discriminate a mechanically “wrong” structure from a stable one particular. The heat maps in More file 1: Figure S1 show that when the stability with the ClusPro structure decreased with time, the stability of your PatchDock’ structure improved via the MD simulation. So it appears unlikely that the PatchDoc’ structure would break up upon a longer MD simulation. 26) “..of Apaf-1 is much more or significantly less evenly negatively charged..” a lot more or significantly less Deleted 27) “..correlation coefficient of 0.9463 as when compared with 0.9558..” how calculated Authors’ response: We’ve got applied UCSF Chimera package [84]. The reference to this software has been added for the Techniques section. 28) Error: “.. Electrostaticpolar interactions or bonds that include salt bridges and prospective H-bonds are Bisphenol A Autophagy commonly considered inside a four cutoff..” the 4A cutoff is for H-bonds. Salt bridges tend to have a cutoff of 8-12A and even longer. The shorter salt bridges in some cases are referred to as H-bonded salt bridges. This also why there needs to be no less than 12A in between the solute as well as the simulation box… Authors’ response: We usually do not see an error here. The criterion for identifying a salt bridge, as initially proposed by Barlow and Thornton [54], is the fact that the distance amongst the heavy atoms of the ionizable groups of charged residues need to be significantly less than four This cut-off of 4 has been used for defining salt bridges in several research, see [503] and references therein, also as within the prior research of cytochrome c interactions with its partners [42]. The cut-off of four was also taken for salt bridges inside 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 towards the revised manuscript. It truly is significant to note that we also go over the long-range interactions. In the original manuscript, we’ve thought of a cut-off of 5 as experimental research show detectable interactions even at this distance [55], furthermore towards the three cut-off used to identify strong hydrogen bonds (Table 3 inside the revised manuscript). To address this comment with the Reviewer, inside the revised manuscript, we’ve added the information that had been collected using a cut-off of 6 to illustrate that any further increase in the MK-7655 Bacterial cut-offShalae.