Er two docking applications did not incorporate energy minimization procedures. The PatchDock’ model was by far the most perturbed, as compared to the outcome from the docking routine, due to the manual editing, which may clarify the pronounced effect of power minimization. 24) I never consider 45 ns can be a lengthy adequate simulation to say something about stability from the complete complex, especially given the enormous size of this complicated. 25) “.. Hence, 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 a lot more careful equilibration of MD is necessary Or that the complexes are wrong Authors’ response: As we’ve got explicitly emphasized inside the revised manuscript, the model structures may be all incorrect, they are just theoretical predictions that await experimental scrutiny. Our process was, having said that, to recognize the residues of Apaf-1 that happen to be Vitamin K2 Data Sheet involved in binding of cytochrome c. We think that we’ve solved this difficulty by combining structural modeling with sequence evaluation. We had to limit our MD simulation time to 45 ns as a result of huge size with the technique. Nevertheless, we consider thatthe simulation time was sufficient to discriminate a mechanically “wrong” structure from a steady 1. The heat maps in Additional file 1: Figure S1 show that though the stability with the ClusPro structure decreased with time, the stability of the PatchDock’ structure enhanced 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 much more or significantly less evenly negatively charged..” extra or much less Deleted 27) “..correlation coefficient of 0.9463 as in comparison to 0.9558..” how calculated Authors’ response: We’ve got employed UCSF Chimera package [84]. The reference to this computer software has been added for the Approaches section. 28) Error: “.. Electrostaticpolar interactions or bonds that involve salt bridges and possible H-bonds are normally considered inside a four cutoff..” the 4A cutoff is for H-bonds. Salt bridges have a tendency to have a cutoff of 8-12A or even longer. The shorter salt bridges at times are named H-bonded salt bridges. This also why there needs to be at the very least 12A among the solute along with the simulation box… Authors’ response: We usually do not see an error right here. The criterion for identifying a salt bridge, as originally proposed by Barlow and Thornton [54], is that the distance amongst the heavy atoms with the ionizable groups of charged residues really should be less than four This cut-off of four has been utilized for defining salt bridges in a lot of research, see [503] and references therein, also as inside the previous studies of cytochrome c interactions with its partners [42]. The cut-off of 4 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 got added the references to all these classical papers for the revised manuscript. It is actually critical to note that we also go over the long-range interactions. Inside the original manuscript, we have considered a cut-off of 5 as experimental studies show detectable interactions even at this distance [55], furthermore for the three cut-off employed to identify strong hydrogen bonds (Table 3 in the revised manuscript). To address this comment of the Reviewer, within the revised manuscript, we’ve added the information that had been collected with a cut-off of six to illustrate that any additional improve within the cut-offShalae.
