Dear Dr. Igaev,

Thank you very much for submitting your manuscript "Microtubule instability driven by longitudinal and lateral strain propagation" for consideration at PLOS Computational Biology.

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Sincerely,

Turkan Haliloglu

Associate Editor

PLOS Computational Biology

Nir Ben-Tal

Deputy Editor

PLOS Computational Biology

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Please see attached file

Reviewer #2: This is an MD-based study on the conformational and energetic consequences of GTP hydrolysis within and across protofilaments in microtubuli, starting from available cryo EM data. The data is very solid, based on long sampling, free energy and entropy calculations, and supports clearly the conclusions, with only one concern with regard to entropy vs free energy considerations, see below. The simulations definitely advance the field and support, by solid numbers, the mechanisms relevant for destabiliziation of MTs.

I fully recommend the study for publication in Plos Comp Biol.

My points:

Entropy calculations and free energy argument Fig. 3: The entropy and free energy calculations both are based on the conformational freedom with regard to dimer spacing (by definition the reaction coordinates in the umbrella sampling), if I am not mistaken. Why is this a good measure, or could there be other changes in free energy involved, such as PF spacing? Also, the NMI and free energies both point into the same direction, but the two fold higher NMI for GDP is used as a stability argument, without referring to the respective dG (which is the actual thermodynamic measure). Couldn’t one read off the free energy ‘penalty’ by confinement (beyond just the entropy) from the free energy maps? Is a mismatch of x causing an increase in free energy of y which is higher (two-fold? or lower?) for GDP compare to GTP? In other words, is the NMI measuring the important thing here?

This question also relates to a major conclusion of the paper: “While not significantly affecting lateral bond stability, the stored elastic energy results in more strongly confined and correlated dynamics of GDP-tubulins, thereby entropically destabilizing the MT lattice.”

It is only entropy? Or could lateral interactions also play a role? This does not seem to be sufficiently quantified by showing that the umbrella sampling and the NMI values point into similar directions.

Given that the difference between GTP and GDP are roughly 0.2nm, it is a bit concerning that the simulation deviate on average from the cryo structures by 0.1nm (Figs 3b,c), luckily both to a similar extent, so that the mismatch remains the same. Still, what could be a reason? This should be discussed.

Minor remarks:

“We hence compared our calculations only with thermal fluctuation experiments because non-elastic deformations as well as induced contact breaking are less likely to occur under such conditions, consistent with our small-strain simulations”

This sentence is misleading. Even if non-elastic deformation are likely in the thermal fluctuation experiments (can not be excluded?), they are essentially at zero strain (or zero-to-small strain) and thus close to your small-strain simulations, that is what you want to say?

Fig 2d: why using the negative stress values and not the positive ones for calculating bending stiffness? Is that common sense? Bending includes both, pulling on one side and pushing on the other, so both elastic moduli (a mean?) could contribute?

“Pure GMPCPP-MTs and GDP-MTs differ in dimer spacing but are homogeneous in their structure and dynamics, because they consist of mechanochemically equal dimers that explore roughly the same conformational space.”

Again misleading: how can the conformational space be the same (of the MTs not only the dimers), but the dimer spacing is different? Would the latte rnot imply different conformational space?

Fig 3b: Please add the meaning of the 0.19nm to caption, just looking at the plots (and searching in the text) eventually resolves it, but it would still help the reader.

Very minor points:

“in a MT” -> in an MT rather?

“father away”

Reviewer #3: This well-written manuscript describes a molecular dynamics study of microtubule energetics. Using recent high-resolution cryo-EM structures, the authors construct effectively infinite models of microtubule filaments in two chemical states corresponding to the initial and the final points of a GTP hydrolysis reaction. Extensive simulations of two protofilament systems revealed a considerable difference in the amplitude of equilibrium fluctuations, indicating a difference in the elasticity of the filament in the two chemical states. The mechanical properties were directly determined by simulating the two systems at various values of the axial pressure tensor. Further analysis of the simulation results produced estimates of the free-energy stored in the filament structure upon GTP hydrolysis, which was found to be in good agreement with the corresponding experimental estimates. The authors next investigate the energetics of filament dimers and trimers through a combination of umbrella sampling simulations and theoretical considerations. The key findings include the free energy cost of lateral incorporation a mismatched tubulin dimer and the degree of correlation in a microtubule lattice.

Overall, this is an exemplary study that demonstrates the power of high-end molecular dynamics simulations in not only examining a qualitative behavior of a biomolecular system but also in providing quantitative estimates of the system’s energetics, including the uncertainty of such determination. While the study is not free from possible artifacts, such as the use of an effectively infinite system for the study of local protein compaction in a dimer filament system, the expected effect of such artifacts is adequately described in the Discussion section of the manuscript. Specific to the microtubule field, the results of the study provide much needed microscopic perspective on the energetics of microtubule growth, setting the stage to future exascale simulations.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Aleksei Aksimentiev

Figure Files:

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Attachments

Attachment

Submitted filename: Igaev_Grubmuller_Review_2020.pdf

Dear Dr. Igaev,

Thank you very much for submitting your manuscript "Microtubule instability driven by longitudinal and lateral strain propagation" for consideration at PLOS Computational Biology. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. 

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Turkan Haliloglu

Associate Editor

PLOS Computational Biology

Nir Ben-Tal

Deputy Editor

PLOS Computational Biology

***********************

A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact ploscompbiol@plos.org immediately:

[LINK]

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Please see attached.

Reviewer #2: My points have been addressed with care. I could not spot the differently colored text in the revised version though

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/ploscompbiol/s/submission-guidelines#loc-materials-and-methods

Attachments

Attachment

Submitted filename: Igaev_Grubmuller_2020_R2.pdf

Dear Dr. Igaev,

We are pleased to inform you that your manuscript 'Microtubule instability driven by longitudinal and lateral strain propagation' has been provisionally accepted for publication in PLOS Computational Biology.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Computational Biology. 

Best regards,

Turkan Haliloglu

Associate Editor

PLOS Computational Biology

Nir Ben-Tal

Deputy Editor

PLOS Computational Biology

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