E location of cytochrome c inside the lobe between the two WD domains. Our modeling procedures aimed at refining the orientation of cytochrome c inside this lobe. Reviewer 2: The approach with the Metolachlor Technical Information authors is very powerful as well as the final model appears to fit-in not merely in the cryoEM density map, but, also is very consistent with current understanding of molecular processes in apoptosome. I wish this article is published because it supplies an opportunity to those working in this region of apoptosome to think about an alternate efficient structural model. Even so authors may perhaps need to think about following points before the attainable publication of this operate: Query 1. It can be not clear in the event the flexibilities connected using the tertiary structures of cytochrome c and Apaf-1 happen to be utilized when authors performed proteinprotein docking making use of different procedures. I believed, at some stage inside the docking (possibly at least within the final stages following the interaction patches are recognized), it is proper to enable some flexibility in the structures with the two associating interfaces.Shalaeva et al. Biology Direct (2015) ten:Page 20 ofobtained in [24], for the PatchDock’ model and also the cryo-EM primarily based structure [PDB:3J2T] [25], respectively, extra clear. We also described the variations in between the fits in extra detail. Question 4. What will be the calculated energies of interaction among the two proteins inside the proposed model and within the model proposed previously Authors’ response: Within the revised manuscript, we provide estimates on the alterations in solvation power of the cytochrome c upon its binding to Apaf-1 (G s) for all model structures that were obtained right after power minimization, also as for the model structure by Yuan et al. [25]; the results are presented in the new Table 2 and discussed.Reviewer’s report three: Dr. Igor N. Berezovsky, Bioinformatics Institute, Agency for Science, Technologies and Study (ASTAR), Singapore 138671, and Department of Biological Sciences, National University of Singapore, Singapore, 117597, Singaporesimultaneously present in the protein and differ according to relevant physiological conditions. MD simulations utilized by authors let 1 to detect dynamic interactions temporal bonds that will be absent within the crystal structure. Although thorough quantitative evaluation of the contribution from bifurcated bonds to protein stability remains to be performed, this work unravels an additional critical aspect of those bonds relevant to protein-protein interactions. Pending experimental verification, part of bifurcated bonds in stability of interfaces can be a precious addition to our understanding with the protein-protein interactions and the mechanisms of their formation and stability. Authors’ response: We are grateful to the Reviewer for these comments and for providing helpful references for the earlier research in the complicated salt bridges hydrogen bonds in proteins. We have incorporated these references into the revised manuscript. We also appreciate the notion that, as outlined by the current terminology for hydrogen bonding “our” complex salt bridges, where one particular donor interacts with two acceptors, ought to be referred to as “double salt bridges” as an alternative to “bifurcated salt bridges”. And still we’ve retained the designation “bifurcated salt bridges” within the revised manuscript because of the following causes. First, the term “double salt bridge” has turn into ambiguous; it is actually also employed to describe a mixture of two pairs of residues forming two “parallel”, easy salt bridg.
