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SUMMARY –

COVID-19 disease is a global pandemic caused by the SARS-CoV-2 virus. Viral protein structures such as Spike, and RNA-dependant RNA polymerase has played a key role in developing structure-guided antibodies and drug designing. Kimberly Dolan and colleagues have made a significant step by unravelling the structure of Membrane (M) protein in a lipid environment by Cryo-EM. M protein plays an essential role in viral assembly and the protein structure shed light on the architecture and features important for protein function. The structure of M protein is very similar to another SARS-CoV-2 protein ORF3a with some key differences that the authors have discussed extensively.

STRENGTHS –

1) Single particle Cryo-EM of small proteins less than 100KDa is a challenging task but the authors were successfully able to stabilize the 50KDa dimeric M protein in lipid nanodisc for structural study.

2) The paper discusses in-depth the structural comparison between the M and ORF3a proteins that are structurally similar but perform different roles in the SARS-Cov-2 virus. While M protein is essential for viral assembly, ORF3a acts as a non-selective calcium channel.

MAJOR CONCERNS –

1) The M protein helps in viral assembly but the paper does not have any in-vitro interaction study with other viral proteins. Interaction study of the M protein with other structural proteins of SARS-CoV-2 would have helped understand the molecular basis of interaction between the structural proteins.                                                                                                                    2) The authors did a comparative structural analysis of the M and ORF-3a but a calcium uptake assay with ORF3a as a positive control could have led to additional experimental data to verify the difference in the function of two structurally homologous proteins.  

3) A recent study by Zhikuan Zhang et al., showed that the M protein overexpressed in mammalian cells purified in GDN showed higher order oligomers ranging from dimeric to hexameric organization. I think that the detergent or the expression system could have affected the oligomerization behavior of M protein in two different conditions. It would be interesting to see if the M protein overexpressed in sf9 cells and purified in GDN shows similar characteristics.

MINOR CONCERNS –

1) The experimental protein structure is compared with the AlphaFold structure but there is no calculated RMSD value assigned to highlight the significant difference between the two structures.

2) In the Method section the FSEC was done on the superose 6 column but the column dimensions are not mentioned. Also in the FSEC Figure S1-(a) the X-axis represents the retention time, which can be modified to elution volume to match the Figures S1-(b),(c) for easy comparison between the FSEC profile with the large-scale size-exclusion profile of M protein in detergent and nanodisc condition.

3) The data processing can be represented as a Cryo-EM processing pipeline to easily track the data processing from the initial micrographs to complete 3D structure and validation.