The 4.4 Å Capsid Structure of the Giant Melbournevirus Belonging to the Marseilleviridae Family

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, Raymond N. Burton-Smith et al. showed the 4.4 Å cryo-electron microscopy structure of the melbournevirus capsid with the block-based reconstruction approach. This manuscript provides valuable structural insights into the melbournevirus capsid and contributes to our understanding of giant virus assembly. However, the manuscript contains a number of inconsistencies in formatting and terminology that should be corrected to ensure clarity and professionalism. These are listed in detail in the reviewer comments.

 

Minor points

1. Line 53: “de novo” should be italic.
2. Line 83: “poly-alanine” should be corrected to “polyalanine”.
3. Lines 101–104: “500 g” and “6,500 g” should be corrected to “500× g” and “6,500× g”, respectively.
4. Line 103: “10-60%” should be corrected to “10–60%”.
5. Line 117: “0.8 – 3.0 μm” should be corrected to “0.8–3.0 μm”.
6. Line 128: “2550 Å” should be corrected to “2,550 Å”.
7. Line 132: “2650 Å” should be corrected to “2,650 Å”.
8. Line 163: “4.8-5 Å” should be corrected to “4.8–5 Å”.
9. Line 180: “these poly-alanine” should be corrected to “these polyalanine”.
10. Line 188: “4.9Å” should be corrected to “4.9 Å”.
11. Line 202: “1250 Å” should be corrected to “1,250 Å”.
12. Line 215: “4.8Å” should be corrected to “4.8 Å”.
13. Line 217: “Figs. S2-S5” should be corrected to “Figs. S2–S5”.
14. Line 227: “Figs. S3-S5” should be corrected to “Figs. S3–S5”.
15. Line 265: “Fig. 3D-E” should be corrected to “Figs. 3D–E”.
16. Line 269: “Fig. 3B, C” should be corrected to “Figs. 3B–C”.
17. Line 269: “Okamoto et al.” should be corrected to “Okamoto et al.”.
18. Line 270: “16kDa” should be “16 kDa”.
19. Line 297: “de novo poly-alanine” should be corrected to “de novo polyalanine”.
20. Line 316: “de novo” should be italic.
21. Line 318: “~6Å” should be corrected to “~6 Å”.
22. Line354: “Pnd” should be “P_nd”.
23. Line 360: “Chihara et al.” should be corrected to “Chihara et al.”.
24. Line 368: “while” should be corrected to “white”.
25. Line 395: “Fig. 3D-E” should be corrected to “Figs. 3D–E”.
26. Line 401: “Fig. S8B-D” should be corrected to “Figs. S8B–D”.
27. Line 404: “Fig. 3B-C” should be corrected to “Figs. 3B–C”.
28. Line 413: “14kDa” should be “14 kDa”.
29. Line 455: “P1d, P2d, P3d” should be “P_1d, P_2d, and P_3d”.
30. Line 485: “Fig. S2-5” should be corrected to “Figs. S2–5”.
31. Line 486: “1MV” should be corrected to “1 MV”.
32. Line 505: “Table S1-S2, Figure S1-S11” should be “Tables S1–S2, Figures S1–S11”.

 

 

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is well-executed and technically sound, representing a parallel method to improve the local resolution of three-dimensional structures of giant viruses capsids. Overall, the work is well written but requires few minor points and further discussion to be considered for acceptance. 

Line 25: The phrase “as previously reported other giant viruses” should be corrected to “as previously reported for other giant viruses.”

Line 33: Please italicize the term Acanthamoeba throughout the manuscript to ensure consistency with standard nomenclature.

Line 99: Replace “PPYG” with PYG

Line 109: Replace “glow-charged” with "glow-discharged"

Lines 191 and 430: Please italicize Marseilleviridae

Discussion: The authors should better discuss the limitations of the block-based reconstruction approach for resolving the 3D structure of giant viruses capsids. While this technique effectively improved local resolution, it still relies on symmetry expansion and local averaging, which may obscure potential capsid heterogeneity. Please discuss this limitation more explicitly in the manuscript.

 

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript describes the 4.4 A structure of the giant virus melbournevirus capsid. It is the third giant virus, after PBCV-1 and ASFV, to have this level of resolution.  I only have a few minor edits.

line 24 ...reported for other...

line 55 virus names are not italicized

line 119 in to is

line 183 ...also processed in the same way ....

line 376 4.4 A should be 3.5 A

line 406 Firstly should be first

line 408 secondly should be second

 

Author Response

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Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript by R. Burton-Smith et al. reports the 4.4 Angstrom Melbournevirus capsid structures using cryo-EM. The authors utilize a similar strategy to the block-based reconstruction, accompanied with the defocus refinement and Ewald’s Sphere correction in RELION, and push the resolution to 4.4 Angstrom, which is the Nyquist limit of their dataset. They identify a unique cup structure on top of each major capsid protein trimer, build a polyalanine model of the penton base protein, and characterize many minor capsid proteins.

Considering the content, I recommend publishing this manuscript in Viruses with minor revisions to some words. Below are some suggestions for the authors.

1. The resolution is indeed being limited by the physical Nyquist frequency. I remember that you have proposed an algorithm called PASR, which can generate super-resolution micrographs from non-super-resolution data. I checked the paper and found that you had also processed this Melbournevirus data with PASR. The resolutions and maps (EMD-37188, 37189, and 37190) were fantastic. After low-pass these maps to 4.0 Angstrom, I can clearly see the structure of the cup. I totally understand that this content belongs to another manuscript, but you might consider include the structure or a prediction based on these improved data.
2. Since you only have around 3100 particles and are trying the block-based reconstruction strategy, you should pick every potential particle, such as the two partially visible particles at top left of Fig. S1A. During 3D classification, you should also avoid removing any particle. After generating sub-particles, doing one-time non-alignment 3D classification will allow to remove the junks and bad sub-particles. By doing so, you may double the number of sub-particles.
3. The boxsize of your block is probably too large if you are trying to push the resolution further. But it doesn’t matter in this dataset since the resolution is already limited by the physical Nyquist frequency.

Comments on the Quality of English Language

There are some grammar errors, which can be easily corrected during the revision.

Line 37-38: “which commonly possesses double-stranded DNA and targeting a wide range of eukaryotic hosts.” It should be “which commonly possess double-stranded DNA and target a wide range of eukaryotic hosts.”

Line 109: “glow-charged” should be “glow-discharged”.

Line 183-184: “Other parts of the mCPs (PC-α and Lattice protein) were also process with the same way ...” It should be “Other parts of the mCPs (PC-α and the Lattice protein) were also processed in the same way...”

Line 314: “named as PC-α”. It should be “named PC-α”

Author Response

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Author Response File: Author Response.docx