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Summary: In this study, researchers used 3D variability analysis (3DVA) combined with atomistic molecular dynamics (MD) simulations to investigate the dynamic motions of human asparagine synthetase (ASNS). By solving the structure of ASNS and performing 3DVA, they suggest that a single side chain's dynamic motion (Arg142) regulates the interconversion between open and closed forms of an intramolecular tunnel. The opening of this tunnel allows for the translocation of ammonia, which is necessary for ASNS’s catalytic function. MD followed up on this initial finding to determine exactly how 

The study highlights the power of cryo-EM in identifying localized conformational changes and demonstrates how conformational dynamics can regulate the function of metabolic enzymes with multiple active sites. However, the lack of experimental electron density shown in the figures (or available publicly) makes it difficult to assess the claims in this study. Additional forward tests of the importance of this blockage via mutagenesis may also uncover why it must be regulated. If this is out of the scope of the current paper, it should be hypothesized and speculated upon in the discussion.

Major Points:

1. In your figures, Please show electron density and all individual atomic positions. This includes Fig. 1d, 2a, and all of Figure 3. 

2. Please show the PCA of the 3DVA. Clarify whether this was done on the entire structure or the tunneling residues. If done on the entire protein, please comment and show if other changes were seen elsewhere.

3. In the RMSD analysis, please clarify what EM coordinates you are using. Are you comparing all structures from the 3DVA? Please also provide raw values as well as normalized values.

4. Your results do not support the claim ‘Our results suggest that changes in the C-terminal active site are propagated over a distance of approximately 20 Å, leading to tunnel opening and ammonia translocation’. While the data here shows that the tunnel can move between an open and closed state in apo form as part of the native fluctuations (revealed by the PCA analysis). No information is presented on how this information is propagated nor how the active site or binding interacts with this motion. Please change the wording of this or explain the mechanism.

Minor Points:

1. Neither the PDB nor the map is publicly available, making it difficult to examine the structures and map independently. Please release them. Also, include information and metrics regarding map sharpening and map-to-model fit. Zenodo is a good option for the 100 structures from the PCA analysis.

2. In Figure 1d, please label the amino acids and chains. Please provide experimental density corresponding to the positions of these residues in this figure or a supplementary figure.

3. In Figure 2a, please provide a legend for what each color represents.

4. How did you determine 5 PCAs for the 3DVA analysis?

5. Please provide details on the normalized RMSF. How was this normalization done?

6. In Figure 4, please provide a legend for all colors of amino acids and tunneling.

7.         Please deposit the coordinate files for the 100 structures used in the 3DVA study and (ideally also the two MD-derived trajectories on Zenodo or a similar repository).

Competing interests

The author declares that they have no competing interests.