Ulating the protein-protein docking operated with rigid bodies (ZDOCK and PatchDock servers) or incorporated only side-chain flexibility (ClusPro). Hence, to refine model structures and to examine the versatile interfaces, we’ve got applied manual editing and energy minimization procedures and, at the final stage, totally free molecular dynamics simulations. We have added the respective clarification towards the Procedures. Query 2. When NBI-31772 manufacturer authors fitted their model inside the cryoEM density map, have they used flexible fitting Use of versatile fitting in the density map is likely to lead to a improved fitting. When flexible fitting is performed, will be the structural interaction attributes proposed by the authors remaining undisturbed Authors’ response: We’ve utilised a rigid fitting procedure as implemented in Chimera application. It could not be excluded that, if applying versatile fitting, we would find yourself having a model structure related to the structure of Yuan and co-workers as shown in Fig. 1a and b and described in [25]; these authors, upon producing their model structure, have employed a sophisticated flexible fitting routine complemented by a manual evaluation. Our additional modest fitting routine has been applied just to demonstrate that our model structure is compatible with all the cryo-EM information. Question three. Just after authors fitted their model in the cryoEM density map, are there any densities inside the zone of cytochrome c and Apaf-1 complex within the map that is unoccupied by any a part of the proposed model Authors’ response: The arrangement on the WD domains of Apaf-1 in our model structure matched completely the arrangement of these domains within the cryo-EM-based model of Yuan et al. [25]. On the other hand, cytochrome c “sits” extra deeply within the PatchDock’ model than in the cryo-EM-based model of Yuan et al. [25]. Within the latter case, cytochrome c is much less deeply buried within the cavity among the two WD domains of Apaf-1, “peeking” slightly out of your estimated electron density (Fig. 1a and b) and, consequently, leaving a part of the electron density map underneath cytochrome c unoccupied. In contrast, the deeper position of cytochrome c in the PatchDock’ model leads to an unoccupied density in the cryoEM map close to the surface in the WD domains (Fig. 1c and d). Within the revised version in the manuscript, we have updated the respective figure by displaying the structural models in two projections (see Fig. 1) to make the distinction amongst the fits from the crystal structures into the electron density map, asReviewer two: Authors of this manuscript are proposing a three-dimensional model for the complex in between cytochrome c and Apaf-1 which consists of WD domain. The basis of generation of this model can be a strategic integration of in depth sequence, structural and evolutionary analyses with molecular dynamics simulations. Amongst the various models initially arrived at, authors favor one of the models that is constant with Cefminox (sodium) Anti-infection identified interaction properties, mutations, conservation of crucial residues and so forth. Interestingly the proposed model is radically distinct from a previously derived model which was determined around the basis of a low-resolution cryoEM map; but, the proposed model too fits rather nicely within the cryoEM density map as reflected by an excellent correlation coefficient. Authors’ response: We thank the reviewer for the comments. We wouldn’t say that our model structure radically differs from the cryo-EM-based model of Yuan and co-workers [24, 25]. In truth, we constructed upon their model, which revealed th.
