Dear Prof. Zhou,

Thank you very much for submitting your manuscript "Binding Free energy Decomposition and Multiple Unbinding Paths of Buried Ligands in a PreQ1 Riboswitch" 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.

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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,

Shi-Jie Chen

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: This is a quite impressive computational study of molecular binding to PreQ1 Riboswitch. The authors studied both the binding interaction and free energies of different ligands to PreQ1, but also used metadynamics to study the unbinding process. The MD simulation is on the micro second time scale, sufficiently long to sample most probable formations. The computational study is comprehensive and thorough and utilized many state-of-the-art technologies. The quality of the paper is high and the manuscript should be published. However, I have the following comments and questions that need to be addressed by the authors.

1. The authors placed Mg++ ions by citing the MCTBI method in [52-55]. Here, the authors should provide a brief explanation of the main point of this method. Also, what are the structural characters of the Mg ions such as metal coordination status.

2. How much does the complex structures used by the authors deviate from the crystal structures?

3. The authors should also list the experimental binding free energies for the cognate and synthetic group (if any) in Table 2 for comparison.

4. The calculated binding free energies for the synthetic group (L1-L3) are mostly positive. How to explain this big discrepancy between calculation and experimental observation? Can this be attributed to the force field used (AMBER ff14sb) or the MM/PBSA method or both? Is it possible that the complex structures for the synthetic group may be to blame?

5. How the Delta S (entropy terms) are calculated?

Reviewer #2: This work uses molecular dynamics simulations to study ligand binding energy and Mg cation interaction of PreQ1 riboswitch aptamer in the apo form and binding with the cognate ligand Q1, Q0 and three synthetic ligands. They conclude that reduction of hydrogen bond donors and acceptors is the main reason for the decreased binding affinities of the synthetic ligands and that synthetic ligands bound state resembles the apo state in several respects. The work provided several important mechanisms of PreQ1 riboswitch’s ligand interaction.

The simulations used in this work provided rich conformation dynamics of PreQ1 riboswitch in apo and in different ligand binding situations. While the current manuscript described certain aspects of conformation changes, it missed large picture of showing and analyzing the connections among the potential important states. For example, even though the eight repeats of simulations are not enough to provide converged Markov State analysis, more examination of similar events, especially in the apo state, would enhance the paper considerably.

Reviewer #3: The work uses atomistic simulations to compare ligand affinity in cognate

vs. sysnthetic riboswitches. Specific nucleotide triplets that contribute

the most to the binding of ligands is identified. Mg++ is fund to be

essential for stabilizing the binding pocket. Specific conclusions on the

binding modes are also made.

Given the importance of riboswitches for gene expression, and as potential

targets for therapeutic, insights into their structure-dynamics-function

relationship are of interest, which makes this work a potentially valuable

contribution. I have two major concerns though:

1. Exactly what are the biological insights/testable predictions? The

authors claim that their work offers new insight for designing riboswitch

ligands. Then, it should be possible to formulate specific, experimentally

testable predictions. After all, these are the main outcomes of theoretical

work.

2. The computational findings are not backed up by comparisons with

experiment, except for one instance (MMPB/SA predicted binding free energy

is found to stronger for the cognate group compared to the synthetic), and

that one comparison is very qualitative. My concern is that readers who

can potentially use the findings -- experimentalists -- may not trust the

findings for lack of specific experimental validation.

2a. A related concern, especially in light o the above,

is the apparent lack of robustness checks: how do the conclusions

depend on details of the methodology, e.g. force-field/ion/water models?

We know that Mg++ is particularly difficult to treat in simulations (see

e.g. works from Aksimetiev's group), and so

is RNA (see Cheatham group works). While duplicating all of the MD

simulations with a different FF/water/ion model maybe a tall order,

checking some of the conclusions against a reasonable alternative to

confirm robustness, is not too difficult.

Other questions/suggestions:

3. page 11 "In comparison, random initial placement of 16 Mg2+ ions was not

effective..." not effective by which metric? Qualitative or quantitative?

4. Methods: How Mg++ ions were treated? There are several, quite different

modern options, including NBFIX and the specific parametrization for nucleic acids

from D. York's group.

4a. Likewise, AMBER 14SB is the protein FF, there are various options in AMBER

to treat RNA.

4b. PME was used with which cutoff?

4c. MMPBSA: which parameters were used? Internal dielectric? Salt

concentration? Probe radius?

5. typos caught:

Abstract: "141 ms" -> 141 microseconds.

page 6: "Because Mg2+ has the same electrons as water," I am sure the

authors mean to say "...the same number of electrons..."

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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

Reviewer #3: None

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Reviewer #1: Yes: John Zhang

Reviewer #2: No

Reviewer #3: No

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Dear Prof. Zhou,

We are pleased to inform you that your manuscript 'Binding Free energy Decomposition and Multiple Unbinding Paths of Buried Ligands in a PreQ1 Riboswitch' 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,

Shi-Jie Chen

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: The responses and the revision of the manuscirpt are satisfactory. It can be accepted now.

Reviewer #2: Thanks for revision. No more comment.

Reviewer #3: The comments were carefully addressed, the paper is in good shape.

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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

Reviewer #3: None

**********

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: Yes: John Zhang

Reviewer #2: No

Reviewer #3: No