Dear Dr. Levy,

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Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data 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 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—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data 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 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—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The paper is about the benchmark of mutation ddG on two software platforms (FEP+ and Gromacs), on 2 protein system.

1. Correlation should not be the main matrix of comparison. RMSE, kendall tau, or AUE (already being used in the results and discussion), can also be used to evaluate the quality of free energy calculations. And Correlation of ddG is not well defined, because of the following reason. There is not really a direction of A to B and B to A, but plotting some ddG edges of A to B or B to A will give different correlation analysis results.

2. In the method section of “FEP calculations using GROMACS”. Please more clearly explain: 1. What soft-core potential is used? Beutler or Gapsys 2. Soft-core potential is applied to both vdw and coulomb or vdw only. 3. “from λ= 0 to λ = 26 …” is confusing and inconsistent with equation 4 in which λ ϵ [0,1]. 4. Are those 27 windows simulated separately or together with Hamiltonian replica exchange. Hamiltonian replica exchange is an available enhanced sampling method in the native Gromacs mdrun. It does not affect simulation through put while bringing in sampling enhancement. If not using, please justify the reason for not using. 5. “AMBER99sb*ILDN” was stated in method, but “Amber99SB-ILDN” was stated in the abstract. 6. How was the charge neutralized when a charge mutation was applied? 7. Which ion parameters were used? 8. Please clarify the exact lambda schedule for example: 0.0, 0.05, … 1.00 for electrostatic … 9. As a benchmark, all the input files (structure, Gromacs mdp files for every steps), analysis files, and results files (e.i. ddG values in csv file) should be provided, either in SI or a openly available repository. Not only should the value of free energy estimation be reported, the uncertainties of each free energy estimation should also be reported.

3. Please make the x-y axis aspect equal when comparing experiment and computed delta G. As 1 kcal/mol in the experiment means the same as 1 kcal/mol in the experiment.

4. Two platforms (FEP+ and gromacs) are using different force fields, enhanced sampling algorithms, and sampling time. From the benchmark results, can we get some insight about the advantages and disadvantages about OPLS4 vs amber99*ildn and REST2 with 16/24 lambda vs 27 lambda with no replica exchange?

5. Does the magnitude of the error correlate with the type of mutation (sides of the amino acid, neutral or charged mutation). Does the error correlate with the place of the mutation (the surface of the protein or in the core of the protein.) With this benchmark, can we get some insight about what kind of mutation is more difficult to calculate with alchemical free energy calculations.

Reviewer #2: This manuscript presents a systematic benchmarking study comparing FEP calculations using Schrödinger FEP+ and GROMACS platforms for predicting mutation-induced stability changes in S. nuclease and T4 lysozyme. The work is methodologically sound and addresses an important gap in the field by directly comparing two widely-used computational platforms. However, several areas require clarification and improvement before publication.

(1)The manuscript reports good correlations between calculated and experimental ΔΔG values. However, I notice that no statistical uncertainties (e.g., standard deviations or confidence intervals) are provided for the computed free energy changes. Given the stochastic nature of molecular dynamics simulations, it would be valuable to assess the statistical reliability of these predictions. Could the authors clarify whether multiple independent simulations were performed for each mutation? If not, it would be feasible to estimate statistical uncertainties using block averaging or bootstrap methods.

(2)The study employs different numbers of λ-windows between the two platforms: 16-24 for Schrödinger and 27 for GROMACS. I am curious about the specific λ-scheduling strategies used in each platform. Could the authors provide details on: (1) the exact distribution of λ values (uniform vs. non-uniform spacing), (2) whether additional λ-windows were placed in critical regions where electrostatic-to-van der Waals transitions occur, and (3) how the different λ-schedules might affect the precision of free energy integration, particularly in regions of poor phase space overlap? Including supplementary data showing dH/dλ distributions or overlap matrices would help readers assess the adequacy of sampling at each λ-window.

(3)I note that Schrödinger (using MBAR) and GROMACS (using BAR) utilized different free energy estimators. While MBAR is theoretically advantageous, its performance often relies on sufficient λ-window sampling. Did the authors evaluate the specific impact of this methodological difference on the comparative results? For instance, would post-processing the GROMACS data with MBAR yield results significantly different from those obtained with BAR?

(4)I notice that the simulation setup differs between platforms regarding the solvent box size: a 5 Å buffer for Schrödinger versus a 10 Å buffer for GROMACS. The authors need to clarify the rationale behind this difference.

(5)The conclusions of the manuscript heavily rely on the convergence of the simulations. Beyond simulation length, the most compelling evidence would be the time-evolution of the free energy for key calculations. The authors need provide convergence plots of ΔΔG vs. simulation time for some representative mutants (e.g., the best and worst predicted cases) to visually demonstrate the convergence and stability of the calculations?

(6) The dataset includes charge-changing mutations, which introduce net charge artifacts under periodic boundary conditions with PME electrostatics—a well-documented source of systematic error in alchemical free energy calculations. I note that the manuscript briefly mentions the use of a "co-alchemical water" approach in Schrödinger to mitigate this issue, but provides no details on its implementation or validation. More critically, there is no discussion of how GROMACS handles net charge corrections for the same mutations. Could the authors clarify: (1) the exact protocol for the co-alchemical water method and whether it fully compensates for finite-size artifacts, (2) whether GROMACS employs any net charge correction schemes (e.g., the analytical correction by Rocklin et al., J. Chem. Theory Comput. 2013, 9, 3072-3083) or simply relies on a neutralizing background plasma, and (3) how these potentially different treatments between platforms might systematically bias the comparative results? This concern is particularly important given the different box sizes noted in point (4), as finite-size effects scale with system size. I would recommend either: (a) a sensitivity analysis quantifying the magnitude of net charge artifacts for a subset of charge-changing mutations, or (b) a thorough literature-based justification demonstrating that the chosen approaches yield negligible systematic errors under the simulation conditions employed.

(7) A fundamental methodological concern is that this study simultaneously compares two different software platforms, two different force fields (OPLS4 in Schrödinger vs. AMBER99SB*ILDN in GROMACS), two different water models (SPC vs. TIP3P), and different enhanced sampling strategies (REST2 in Schrödinger vs. standard MD in GROMACS). This multifactorial design makes it challenging to disentangle whether the observed differences in performance arise from: (1) software implementation details, (2) force field parameterization, (3) water model effects, or (4) sampling methodology. While I recognize that systematically testing all possible combinations may be beyond the scope of this benchmarking study, this confounding factor represents a significant limitation that should be explicitly acknowledged in both the Discussion and Limitations sections.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes:Chenggong HuiChenggong HuiChenggong HuiChenggong Hui

Reviewer #2: No

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Benchmarking Free Energy Calculations: Analysis of Single and Double Mutations Across Two Simulation Software Platforms for Two Protein Systems

PONE-D-25-56221R1

Dear Dr. Levy,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Yong Wang

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data 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 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—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data 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 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—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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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: All the questions have been addressed.

Reviewer #2: (No Response)

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #1: Yes:Chenggong HuiChenggong HuiChenggong HuiChenggong Hui

Reviewer #2: No

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