Ulating the protein-protein docking operated with rigid bodies (ZDOCK and PatchDock servers) or incorporated only side-chain flexibility (ClusPro). For that reason, to refine model structures and to examine the flexible interfaces, we’ve applied manual editing and power minimization procedures and, in the final stage, cost-free molecular dynamics simulations. We’ve got added the respective clarification to the Techniques. Query two. When authors fitted their model in the cryoEM density map, have they applied flexible fitting Use of versatile fitting in the density map is most likely to result in a greater fitting. When versatile fitting is accomplished, are the structural interaction capabilities proposed by the authors remaining undisturbed Authors’ response: We have applied a rigid fitting procedure as implemented in Chimera software. It could not be excluded that, if applying flexible fitting, we would wind up using a model structure similar to the structure of Yuan and co-workers as shown in Fig. 1a and b and described in [25]; these authors, upon creating their model structure, have applied a sophisticated versatile fitting routine complemented by a manual evaluation. Our much more modest fitting routine has been applied just to demonstrate that our model structure is compatible together with the cryo-EM information. Question 3. Following authors fitted their model inside the cryoEM density map, are there any densities inside the zone of cytochrome c and Apaf-1 complicated inside the map that may be unoccupied by any a part of the proposed model Authors’ response: The arrangement with the WD domains of Apaf-1 in our model structure matched completely the arrangement of these domains inside the cryo-EM-based model of Yuan et al. [25]. On the other hand, cytochrome c “sits” extra deeply inside the PatchDock’ model than inside the cryo-EM-based model of Yuan et al. [25]. Inside the latter case, cytochrome c is less deeply buried inside the cavity in between the two WD domains of Apaf-1, “peeking” slightly out from the estimated electron density (Fig. 1a and b) and, consequently, leaving a a part of the electron density map underneath cytochrome c unoccupied. In contrast, the deeper position of cytochrome c inside the PatchDock’ model results in an unoccupied density inside the cryoEM map close for the surface of the WD domains (Fig. 1c and d). In the revised version of your manuscript, we’ve got updated the respective figure by displaying the structural models in two projections (see Fig. 1) to create the difference in between the fits on the crystal structures into the electron density map, asReviewer 2: Authors of this manuscript are proposing a three-dimensional model for the complex among cytochrome c and Apaf-1 which includes WD domain. The basis of generation of this model is a strategic integration of comprehensive sequence, structural and L-5,6,7,8-Tetrahydrofolic acid Description evolutionary analyses with molecular dynamics simulations. Amongst the many models initially arrived at, authors favor one of the models which is consistent with known interaction properties, mutations, conservation of crucial residues and so on. 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 also fits pretty Ristomycin Antibiotic nicely within the cryoEM density map as reflected by a superb correlation coefficient. Authors’ response: We thank the reviewer for the comments. We would not say that our model structure radically differs in the cryo-EM-based model of Yuan and co-workers [24, 25]. Actually, we constructed upon their model, which revealed th.
