The author used Kinematic flexibility analysis (KFA) method to investigate the conformational flexibility of SARS-CoV-2 main protease (Mpro) which is considered a promising drug target. They analyzed the effect of mutations for 47 mutation sites on the conformational flexibility of Mpro, using 69 crystal structures of Mpro that are bound to different ligands.
The results of this study can be useful for the antiviral drug development against SARS and probably deserve to be published. However, I have concerns regarding the manuscript, some of which I detail below. Overall, I don’t think it should be published in its current form without extensive revisions.
1) The manuscript requires significant editing. There are many grammar and spelling mistakes. Moreover, the paper is hard to read, and the wording and nomenclature used throughout are not consistent with the field.
Some examples:
• The author used Sars Covid19 when they discuss the coronavirus in the manuscript. Sars Covid19 (The Coronavirus disease-19 or COVID-19) is a disease caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). The author should use SARS-CoV-2 when they discuss the coronavirus.
• The author used “hydrogen binding number”. They should use the “number of hydrogen bonds”.
• The author used “have more (or less) binding hydrogen bonds”. They should use “have more (or less) hydrogen bonds between protein and ligand”.

2) The authors make several strong claims in the introduction without any adequate citation, and they are not relevant to the scope of their work.
Such as:
“which caused a worldwide panic. It seriously blocked our education, production, and lifestyle.”

3) The authors present their results, but they do not discuss their findings.
• The authors should add a discussion of their results and importantly, compare it to other studies that looked at dynamics. A few methods which predict the effects of mutations on protein stability are: Dynamut2 (Rodrigues et.al. 2018) or SDM (Worth et. al. 2011, Pandurangan et. al. 2017)
• They found that P99L mutation causes the crystal structures to be much more flexible. They should discuss why and compare this conclusion to the findings of other studies.
• They found that 8 crystal structures have a larger ratio of rigidified DoFs to cycle DoFs after the single mutation so that means these 8 mutants are more stable than WT? Did they investigate these structures in terms of dynamics? What could be the underlying reason?
• Some of the ligands bind covalently to Cys145 in protein-ligand complexes. These ligands might have partially/completely modified MPro. How does this affect the authors’ analysis?

4) The author should mention the protein preparation method that they used and how they assign the protonation states.

5) They analyzed 112 Mpro crystal structures in their previous paper (Xiyu Chen et al. 2022) while they investigated 69 crystal structures in this study. Why did they exclude the rest of the structures?

6) Additional comments to improve the manuscript:
• The author used ‘between no mutation to mutation’. They should use terms like ‘wild-type (WT) and mutated structures (or Mpro variants or mutants etc.)
• The author should use ‘single residue mutation or substitution’ instead of “single mutation”.
• The author used ‘Mpro crystal structures binding with different ligands’ throughout the manuscript. They can use terms like the Mpro–ligand complexes.
• What do the different colors refer to in Figures (7-21) which represent molecular visualizations of Mpro crystal structures? Why there are some red circles for some structures?
• In Figure 6(a), the authors show a scatter plot of the ratio of rigidified DoFs to cycle DoFs for mutant vs WT structure but they used “mutation vs no mutation” as x and y-labels. The axis labels throughout the manuscript should be clear about what they refer to.
• Page 10 Figure 3B. The author should use a different color for the linker loop which is currently red as if as a part of Domain II.
• Page 10 Line 10. “These mutation positions:” should be “The residues undergoing mutations: A7, …” or “The mutations are: A7V, …”