Effect of alpha-tubulin acetylation on the doublet microtubule structure

Joint Public Review:

Summary:
The study "Effect of alpha-tubulin acetylation on the doublet microtubule structure" by S. Yang et al employs a multi-disciplinary approach, including cryo-electron microscopy (cryo-EM), molecular dynamics, and mass spectrometry, to investigate the impact of α-tubulin acetylation at the lysine 40 residue (αK40) on the structure and stability of doublet microtubules in cilia. The work reveals that αK40 acetylation exerts a small-scale, but significant, effect by influencing the lateral rotational angle of the microtubules, thereby affecting their stability. Additionally, the study provided an explanation of the relationship between αK40 acetylation and phosphorylation within cilia, despite that the details still remain elusive. Overall, these findings contribute to our understanding of how post-translational modifications can influence the structure, composition, stability, and functional properties of important cellular components like cilia.

Strengths:
1. Multi-Disciplinary Approach: The study employs a robust combination of cryo-electron microscopy (cryo-EM), molecular dynamics, and mass spectrometry, providing a comprehensive analysis of the subject matter.
2. Significant Findings: The paper successfully demonstrates the impact of αK40 acetylation on the lateral rotational angles between protofilaments (inter-PF angles) of doublet microtubules in cilia, thereby affecting their stability. This adds valuable insights into the role of post-translational modifications in cellular components.
3. Exploration of Acetylation-Phosphorylation Relationship: The study also delves into the relationship between αK40 acetylation and phosphorylation within cilia, contributing to a broader understanding of post-translational modifications.
4. High-quality data: The authors are cryo-EM experts in the field and the data quality presented in the manuscript is excellent.
5. Depth of analysis: The authors analyzed the effects of αK40 acetylation in excellent depth which significantly improved our understanding of this system.

Weaknesses:
I have no major concerns about this paper, but would recommend that a few minor issues be addressed.

1. Lack of Statistical Details: The review points out that the paper could benefit from providing more statistical details, such as the number of particles and maps used for analysis, randomization methods, and dataset splitting for statistical analyses.
2. Questionable Conclusion Regarding MIPs: The reviewer suggests caution in the paper's conclusion that "Acetylation of αK40 does not affect tubulin and MIPs." The reviewer recommends that this conclusion be more specific or supported by additional evidence to exclude all other possibilities.
3. Need for Additional Visual Data: The reviewer recommends that an enlarged local density map along with fitted PDB models be provided in a supplementary figure, such as Figure 4.

Overall, the paper is strong in its scientific approach and findings but could benefit from additional statistical rigor and clarification of certain conclusions.