Comparative Study of High-Resolution LysB29(Nε-myristoyl) des(B30) Insulin Structures Display Novel Dynamic Causal Interrelations in Monomeric-Dimeric Motions

Round 1

Reviewer 1 Report

The manuscript "Comparative Study of High-resolution LysB29(Nε-myristoyl) des(B30) Insulin Structures Display Novel Dynamic Causal Interrelations in Monomeric-Dimeric Motions" by Ayan et al., describes the crystallographic structure of commercially branded insulin Detemir to 1.7 A resolution.

Although this Reviewer recognises the effort put in this work, and anticipates the potential scientific input into the fiel, the manuscript is very hard to follow due to the way it is written. The "monomer" wording confounds itself with proper monomers, as well as the dimer, dimer of dimers, hexamer and so on, description is confusing.

This Reviewer is high thankful to the Authors for providing the PDB's; this is not a common practice and this Reviewer acknowledges the confidence shown by providing this. However, the maps on Figure 1C, i, ii, iii and iv, are high puzzling since the fitting seems not to be supported by the electron density. Moreover, it is not clear why the authors decided to truncate the data to 1.7  while having a 3.28 value for I/sigI in the highest resolution shell. This Reviewer urges the Authors to revisit data processing, extend resolution and use STARANISO for data scaling. This approach might improve significantly the data/resolution and, hence, the results/conclusions derived (and, ultimately, the manuscript itself).

Finally, it is not clear (maybe this Reviewer did not get it), how the ellipsoid analysis was performed, since (apparently), anisotropic data processing was not performed. Are these simply derived from TLS analysis?

This Reviewer kindly asks the Authors to take this comments as constructive and, hopefully, wishes that these might be used to improve the manuscript. The Reviewer wishes the Authors all the best for their future endeavours.

Author Response

Dear Crystals Editorial Office,

The response letter is completed and submitted in PDF format.

Sincerely,

 

Author Response File: Author Response.pdf

Reviewer 2 Report

In this contribution by Ayan et al., the high resolution structure of insulin detemir is reported together with the GNM analysis of this structure. 

The manuscript requires only minor language adjustments but some scientific issues should be resolved before the acceptance. 

First of all, I do not really understand that much of the focus is spent on the unit cell origin - it is somewhat obvious as the protein was crystallized in a different space group, so I don't think this is a very relevant part of the manuscript. I agree it also dictates the packing and the arrangement of molecules seems different, but it would be more relevant if such an arrangement would have been also observed in solution. 

What was the reason the data cut-off is at 1.7 Å resolution? All indicators in the table 1 clearly show that the data extends well beyond that value.

The lines 90-92 state that the models were renumbered, what was the rationale???

In figure 1B it is impossible to see anything as it is too crowded. Densities for MYR look somewhat strange. What is the level of the density? Also the authors should provide images with omit map and not 2fo-fc.
In figure 3 - the normalised B-factors are shown, but the process of normalisation is not described. 

In lines 226-231 the radiation damage is discussed but the link with the rest of analysis is not very obvious. Also the comparison of radiation damage between two structures is meaningless as the information about the dose etc. is missing. 

Author Response

Dear Crystals Editorial Office,

The response letter is completed and submitted in PDF format.

Sincerely,

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript "Comparative Study of High-resolution LysB29(Nε-myristoyl) des(B30) Insulin Structures Display Novel Dynamic Causal Interrelations in Monomeric-Dimeric Motions" by Ayan et al., describes the crystallographic structure of commercially branded insulin Detemir. This is the second revision made by this Reviewer to the present manuscript.

Although this Reviewer recognises the effort put in this work, and the attempt made to answer this Reviewer's comments/concerns, this Reviewer does not feel confident accepting this manuscript. Not only the comments/concerns were not properly addressed, but the new data processing raises several new questions.

This Reviewer will try to illustrate the serious concerns he has with this work:

1. When this Reviewer pointed out the fact that the Results section regarding the protomer assembly was hard to follow, this Reviewer was expecting some rewording and rephrasing of the next; to this Reviewer surprise, the Authors do not do any of this, but rather point to the paragraph where that assembly is described;

2. This Reviewer raised concern on the maps on Figure 1C, i, ii, iii and iv, regarding the MYR moietes, which are highly puzzling since the fitting seems not to be supported by the electron density; to this Reviewer surprise, nothing was done about this, but just making themselves available to calculate an OMIT map in PHENIX; probably this should have already been done, clearly reassuring this Reviewer that indeed those moieties were properly moddelled;

3. Moreover, how can the data have been reprocessed, but the electron densities in the above mentione figure/pannels look exactly the same? In addition, a CC1/2 of 20% is below the common practice and SOP's in the field (check Curr Opin Struct Biol, . 2015 Oct;34:60-8. doi: 10.1016/j.sbi.2015.07.003. Epub 2015 Jul 24).

For this, my recommendation is to Reject the manuscript, while urging the Authors to take time and a (serious) effort to address this issues.

Author Response

Dear Madam/Sir,

You can find the response letter in the attachment.

Sincerely,

Author Response File: Author Response.docx