Dear Dr. Hagan,

Thank you very much for submitting your manuscript "Self-Assembly Coupled to Liquid-Liquid Phase Separation" 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. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

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

[1] A letter containing a detailed list of your responses to the 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).

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Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. 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,

Huan-Xiang Zhou

Guest Editor

PLOS Computational Biology

Arne Elofsson

Section 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: Comments are attached.

Reviewer #2: This is an interesting theoretical development that addresses the thermodynamic and dynamic ramifications for the modulation of viral-capsid-like assembly in cellular compartments that maintain a higher concentration of subunits. The results are suitable for publication in PLoS CB, but revision is necessary to clarify/address the following issues:

1. The authors elect to use “domain” to describe the compartment that maintain a higher concentration of subunits. This term is not ideal if the authors aim to frame their investigation in the context of recent advances in the study of biomolecular condensates (which is clearly the case judging from the large number of references in this area that the authors included). These condensates are usually referred to as “compartments”. In this very field that the authors aim to contextualize their work, “domain” usually refers to protein domains. So the authors’ terminology will not help facilitate understanding by readers who are familiar with the biomolecular condensate terminology. I suggest that the authors to change “domain” to “compartment” (they did use “compartmentalization” on p.12 of the manuscript). Failing that, they should at least state clearly at the outset that their “domain” is equivalent to intracellular membraneless biomolecular compartments.

2. Though it is not stated explicitly in the text, the formulation in Eqs.13-14 indicates that subunits can only be added one-by-one in the authors’ model. In other words, associations of, say, two n=2 complexes is postulated to be impossible (because there are no such terms in Eqs.13-14). Is this the case? If so, this limitation has to be justified. Does it have an experimental basis in viral capsid assembly? Irrespective of whether viral capsid assembly obeys this rule, the authors should discuss (and/or show additional calculation) what will happen when this limitation is lifted.

3. Notation problem: fN is used to denote fraction of completed assembly in Fig.2 and Fig.3 but is also used (on the same page) to represent associate rate constant for the n=N state in Eq.14. This situation should be rectified. Authors should use different symbols for different quantities in the same paper, especially when different meanings of the same symbols are invoked so close to each other in the text.

4. Additional information should be provided in Fig.3 to show corresponding data for ρT > 1μM (by adding a figure or adding parts to this figure or simply by adding curves to the existing figure). This is important for illustrating the decreases seen for the red curves in Fig.2 for larger ρT , which is an interesting finding. To make this correspondence clearer, vertical dashed lines should be added to indicate t = 1 day time point (24 hr) along the horizontal scales of these plots.

5. It is important to emphasize that the kinetic traps mentioned in the present work are caused by depletion of subunits but not “malformed assembly” (mentioned in page 12). Malformed assemblies can also cause kinetic traps (as their disassembly requires an energetic cost). This matter should be commented upon with a clarification about the basic difference between these two types of kinetic traps.

6. More specific examples should be provided in the concluding discussion about what real biophysical process this formulation is expected to be applicable to. What kind of viral capsid? Which intracellular compartments (domains) are the likely locations for viral capsid assembly?

7. The authors clearly aim to be rather comprehensive in their citing literature in the Introduction. In this regard, references for several key advances in the theoretical study of biomolecular condensates are missing, which should be included in the revised version of this manuscript, as follows: Lin et al., Phys Rev Lett 117:178101 (2016) (first theory for sequence-dependent biomolecular phase separation ) should be included around ref.[50], and Lin & Chan, Biophys J 112:2043-2046 (2017); Amin et al., J Phys Chem B 124:6709-6720 (2020) – two theoretical works that address the relationship between single-chain and two-chain association properties and multiple-chain phase separation propensity – should be included around ref.[63].

8. Likely typographical errors:

(i) 3rd line below Eq.10: “Kdom → 0” should be “Kdom → 1”?

(ii) Eq.A3: subscript n is missing in the two diffusion coefficient. Judging from these typos, there are likely others – this referee did not do an exhaustive check. The authors are strongly encouraged to double-check to make sure that their notation is clear and consistent and without omissions and typos.

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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: Yes

Reviewer #2: Yes

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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.

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Attachments

Attachment

Submitted filename: 2022-Dec-PlosComputBiol.pdf

Dear Dr. Hagan,

Thank you very much for submitting your manuscript "Self-Assembly Coupled to Liquid-Liquid Phase Separation" 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 just update the colors in Fig 1.

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 just update the colors in Fig 1.

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: The authors have made a very good attempt at addressing the comments raised in the first review. Their explanations are more thorough and the changes have significantly improved the clarity and scope of the paper. One additional comment pertains to figure 1; there, the color scheme used for the text (i.e., on the compartment) makes for low readability. The authors should consider a different color palette as would make the figure more accessible.

Reviewer #2: The authors have adequately (in fact quite thoroughly) addressed my previous concerns. I recommend publication of the current revised version of this manuscript.

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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: 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: Yes: Hue Sun Chan

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. Hagan,

We are pleased to inform you that your manuscript 'Self-Assembly Coupled to Liquid-Liquid Phase Separation' 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,

Huan-Xiang Zhou

Guest Editor

PLOS Computational Biology

Arne Elofsson

Section Editor

PLOS Computational Biology

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