In response to the Comment by Rupp and Segelke concerning our paper published in Nature Structural Biology last year, we apologize for omitting that the occupancy of the peptide is estimated to be ∼30–40%. Our published study was a rapid soak freeze-trap experiment, and in order for us to observe the product bound form of the peptide, it is not unexpected that the peptide is of lower occupancy in the crystal than the protein molecule. However, we should have noted this in our publication. The stereochemical quality of the peptide is low, and we believe this to be due to the occupancy of the peptide. However, since the peptide is disordered in solution, we did not want to constrain the peptide to an artificially high quality stereochemical model. Attempts to obtain higher occupancy binding of the peptide were not successful. The peptide is most clearly observed when one compares the active site of both the apo (1f82.pdb, r1f82sf.ent) and peptide bound (1f83.pdb, r1f83sf.ent) forms. This latter comparison helps to resolve any potential phase bias that might have occurred from the molecular replacement solution using the holotoxin model.

More germane to the science is the discovery that the synaptobrevin peptide binds in the same location as the translocation domain of the holo-botulinum neurotoxin after translocation domain separation. This observation is consistent with published biochemical studies as cited in our publication and it provides a potential explanation for previously unexplained observations in the field of botulinum neurotoxin research. Additional biochemical data are accumulating that support our observation and I suspect we will read more articles in the near future on this topic. Based on our 2.2 Å apo and 2.0 Å substrate-bound structures and additional structural studies that we have conducted since publication, we continue to support our published proposal on how the neurotoxin cleaves synaptic vesicle proteins. However, our goal is to accurately understand how the neurotoxin works and if our proposal is not correct, we would like to know as soon as possible to advance the field in a forward direction. We look forward to seeing this matter resolved in the peer-reviewed literature in the near future.

In summary, we thank Rupp and Segelke for pointing out that we did not state the occupancy of the peptide in our published article, and we apologize to the community for this omission.