Dear Dr. Seoane,

Thank you very much for submitting your manuscript "Soft disorder modulates the assembly path of protein complexes" 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 make sure to also address the concerns from Reviewer 1: The topic is interesting, and the conclusions potentially interesting. The only concern I have is that the IDR based interactions often happen with high on/off rates, and I am not sure to what extent they are involved in building up large complexes. The authors might want to comment on this in the discussion.

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,

Rune Linding

Guest Editor

PLOS Computational Biology

Arne Elofsson

Deputy Editor

PLOS Computational Biology

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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]

Please make sure to also address the concerns from Reviewer 1: The topic is interesting, and the conclusions potentially interesting. The only concern I have is that the IDR based interactions often happen with high on/off rates, and I am not sure to what extent they are involved in building up large complexes. The authors might want to comment on this in the discussion.

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: The topic is interesting, and the conclusions potentially interesting. The only concern I have is that the IDR based interactions often happen with high on/off rates, and I am not sure to what extent they are involved in building up large complexes. The authors might want to comment on this in the discussion.

Reviewer #2: In this manuscript the authors study the role of protein flexibility in protein complex assembly. The work presented here follows from a previous study from the same authors (Seoane et al. PLOS Comp Bio 2021) where they have shown some degree of association between protein flexibility , estimated by a normalised B-factor, and interface residues. In the previous publication, these authors hinted at the possibility that flexibility could be predictive of interface regions along a path of assembly from monomeric to larger assemblies. This suggestion is now followed up statistically in the current manuscript. Essentially, the single point of the manuscript is that interface residues of complexes having larger numbers of subunits can be somewhat predicted by the flexibility of residues in complexes having lower number of subunits. This result is shown in figure 5 with figures 6,7 and 8 essentially trying to show that the result is robust to different ways of looking at the data. More broadly the relation between interface residues and flexibility has a very long history of study with quite many perspectives, including ideas around folding upon binding; flexibility of core vs. periphery of interface residues; and changes in entropy upon binding among other ideas and literature. The work done here, where the authors study changes in flexibility in a given protein with increasing numbers of bound interactors is a useful addition to the literature.

I only have a small number of concerns and they are primarily related with presentation and clarity instead of technical issues:

1 - The biggest issue I have had while reviewing the paper was the clarity of the manuscript itself. There is essentially one main result in the paper but there are 9 figures with the fist real result starting in figure 5. Figures 1 to 4 are diagrams and a non critical result figure. Figures 6, 7 and 8 are controls and figure 9 is a very short result related with Alphafold predictions that probably should be removed. The text itself is also very difficult to follow because the authors introduce many acronyms and symbols (SDR, IR,k, K, PPV, etc) and some paragraphs are really full with acronyms and sentences one needs to re-read a few times to understand. It would be great if the authors could simplify and improve on the readability of the work.

1.1 – I suggest the authors could simplify and merge figures 1, 2 and 3 and move figure 4 to supplementary.

1.2 – I don’t really see the point of adding the Alphafold result at the end of the manuscript. It is a small example and as the authors state, it would require a proper analysis. I don’t see how it adds to the central claim of the work. It would make more sense to develop into a proper stand alone piece of work. Instead, the authors could leave some of these ideas in the discussion section alone without any specific results.

1.3 – There are some parts of the results that could be moved to the methods section. Page 5 and 6 – the definitions of how the DAG was built could be, at least in part, moved to the methods. Page 9, around lines 202-206, the definition of random might be better in the methods.

1.4 – It is hard to say what to do with the acronyms but, at least in places, the authors could try to avoid using them to make it easier for readers that are not as familiar with their terms.

2 – One point that stood out from a technical perspective was that the authors have analysed the results in the form of how often the TPR and FPR are better than random. One of the key result is in Figure 5A where the authors report that 74% of the predictions (i.e. flexible regions predicting the next interface region) are better than random. While this seems to be the case I think it is also noteworthy that for the majority of these cases the predictions are really just somewhat better than random. For a given FPR most of the predictions have a TPR that is quite close but above random. Overall, this association reported in the manuscript seems real but having a small effect size. I think it would be important to comment on this in the discussion section.

3 – The authors make a big point, both here and in the previous manuscript about the term “soft disorder”. I don’t really see the need to come up with a new term, why not just use the term flexibility ? Or relative flexibility since the authors used a protein normalized metric.

Additional minor suggestions:

- It is not common to underline words in scientific communications, might be better to remove this

- The abstract usually opens with some form of introductory sentence(s) as to why the work/topic is important.

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Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data and code should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: None

Reviewer #2: Yes

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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, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

Review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.

If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr. Seoane,

Thank you very much for submitting your manuscript "Soft disorder modulates the assembly path of protein complexes" 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 fix the repeated paragraphs

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,

Arne Elofsson

Section Editor

PLOS Computational Biology

Arne Elofsson

Section 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:

Please fix the repeated paragraphs

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: The manuscript has been improved and the main message is now easier to get. Thank you.

Reviewer #2: The authors have addressed my previous concerns, in particular by making the manuscript more accessible to others and by adding a small note in the discussion about the small effect size of the effect they are describing here. Not exactly those words but at least it is mentioned.

In the PDF version I read there is a repeated paragraph in line 203 starting with "By construction .."

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Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data and code should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: None

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, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

Review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.

If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr. Seoane,

We are pleased to inform you that your manuscript 'Soft disorder modulates the assembly path of protein complexes' 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,

Arne Elofsson

Section Editor

PLOS Computational Biology

Arne Elofsson

Section Editor

PLOS Computational Biology

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