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Thank you very much for addressing all concerns of the reviewers and for careful revision.
# PeerJ Staff Note - this decision was reviewed and approved by a PeerJ Section Editor covering this Section #
Please address the issues raised by all three reviewers.
The paper is generally well written, with only a few minor errors that need to be corrected. Literature cited properly. Figures are good, although the main difference compared to an existing structure of HC/FA, namely the presence of the electron density for the loop 1261-1268 is not illustrated.
Nothing novel in the presented results compared to those already available in the literature, but the experiments have been done well.
Data are sound, conclusions well stated, although nothing novel has been presented.
The main difference between the structures described here, namely the visibility of the loop 1261-1268 in HC/FA, is of only limited interest, since conformations of flexible loops, even those involved in biologically relevant interactions, are not very illuminating unless they are seen in a proper context, and not as crystal contacts. Similarly, the presence or absence of disulphides is also simply related to conditions of crystallization.
A few minor errors that should be corrected:
Lines 32-33 "The toxins target...through a dual receptor binding model". No, they don't do it through a model, but through binding to two receptors. Please rephrase.
Line 37 "A through to /G" - please make it "A through /G"
Line 115 remove comma after "group,"
Line 159 is "invaluable" the best word here?
Legend to Fig. 1 5MK1 is probably in error, 5mk6 meant
The manuscript is well written.
The structural analysis is overall robust, although some additional information about the findings is requested as described in the general comments.
The manuscript describes the high resolution crystal structures of the receptor binding domain (Hc) from two botulinum neurotoxins (BoNTs), Hc/FA and Hc/A1. These structures can potentially provide insights into the binding specificity of these BoNTs in recognizing particular receptors. The main new findings include the observation of an Hc/FA loop conformation that was disordered in a previous structure, and the discovery of a disulfide bond in both Hc/FA and Hc/A1 that was not observed in previous structures. The structural data are technically sound. However, multiple structures of both Hc/FA and Hc/A1 had been previously determined either for the domains themselves or as part of the whole protein. Therefore there need to be more discussion of the new scientific insights gained from these structures, in comparison to the previous structures, for both Hc/FA and Hc/A1.
1) One of the main new findings is the R1261-R1268 loop conformation in Hc/FA, yet there is no figure showing its electron density or relating this loop to receptor binding (a role suggested by the authors) by comparing the current structure to previous complex structures.
2) In Fig. 1, it will be useful if the authors could show the locations of the disulfide bonds and the R1261-R1268 loop. In addition, it may be worth indicating the N and C-termini on the structure.
3) Table 1, the Rwork and Rfree of the 1.45 Angstrom resolution structure seem high, relative to its resolution. It will be informative if the authors can comment on this, particularly compared with the other two lower resolution structures. Did the refinement use anisotropic B factors?
4) line 134, the average B factor of the loop should be provided.
The manuscript is well written and easy to follow.
The experiments are well designed and well performed. The structure of the receptor binding domain from BoNT/FA has been reported before. The authors do however provide new insight and clearly reference and compare to the previous study. The structure of HcA has been reported, however an interesting difference in a disulphide bond and the resulting structural rearrangements is reported.
All statistics look good and the data is of good quality.
This is a solid study reporting a some new observations. The paper is well written and should be accepted after minor revisions.
The contouring level and type of map (Fo-Fc?) for figure 2 should be added. A description of how the map was calculated should be added to material and methods (what program was used).
P422 is an unusual space group. Alternative space groups should be examined and the possibility of twinning should be considered (could give false high symmetry).
A sequence alignment between subtypes and serotypes should be included to visualise and discus the conservation of the cysteine.
The position of the cysteine and the loop that is discussed, should be indicated in the figure 1.
A picture shoving an overlay with the ganglioside complex should be included to clarify the role of the loop in BoNT/HA that is discussed.
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