Er two docking applications didn’t incorporate power minimization procedures. The PatchDock’ model was the most perturbed, as when compared with the outcome with the docking routine, due to the manual editing, which might clarify the pronounced impact of power minimization. 24) I don’t assume 45 ns is really a lengthy adequate simulation to say anything about stability with the entire complicated, especially provided the massive 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 through the MD simulation..” this worries me. Could it be that a far more careful equilibration of MD is required Or that the complexes are incorrect Authors’ response: As we’ve explicitly emphasized inside the revised manuscript, the model structures might be all wrong, they’re just theoretical predictions that await experimental scrutiny. Our activity was, even so, to identify the residues of Apaf-1 which can be involved in binding of cytochrome c. We think that we have solved this dilemma by combining structural modeling with sequence analysis. We had to limit our MD simulation time to 45 ns as a result of large size of the system. Still, we assume thatthe simulation time was enough to discriminate a mechanically “wrong” structure from a steady 1. The heat maps in Added file 1: Figure S1 show that when the stability of your ClusPro structure decreased with time, the stability of the PatchDock’ structure increased by way of the MD simulation. So it seems unlikely that the PatchDoc’ structure would break up upon a longer MD simulation. 26) “..of Apaf-1 is additional or much 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’ve made use of UCSF Chimera Ethoxyacetic acid MedChemExpress package [84]. The reference to this software has been added to the Strategies section. 28) Error: “.. Electrostaticpolar interactions or bonds that involve salt bridges and potential H-bonds are frequently regarded within a four cutoff..” the 4A cutoff is for H-bonds. Salt bridges have a tendency to have a cutoff of 8-12A and even longer. The shorter salt bridges sometimes are named H-bonded salt bridges. This also why there need to be a minimum of 12A involving the solute and the simulation box… Authors’ response: We usually do not see an error here. The criterion for identifying a salt bridge, as originally proposed by Barlow and Thornton [54], is the fact that the distance between the heavy atoms on the ionizable groups of charged residues needs to be much less than four This cut-off of four has been utilized for defining salt bridges in quite a few studies, see [503] and references therein, too as in the preceding research of cytochrome c interactions with its partners [42]. The cut-off of four was also taken for salt bridges in 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 can be critical to note that we also discuss the long-range interactions. Within the original manuscript, we’ve deemed a cut-off of 5 as experimental research show detectable interactions even at this distance [55], in Ozagrel web addition to the three cut-off used to identify powerful hydrogen bonds (Table 3 within the revised manuscript). To address this comment from the Reviewer, inside the revised manuscript, we have added the information that had been collected having a cut-off of 6 to illustrate that any further enhance in the cut-offShalae.
