Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs

  1. David M. Kern
  2. Ben Sorum
  3. Sonali S. Mali
  4. Christopher M. Hoel
  5. Savitha Sridharan
  6. Jonathan P. Remis
  7. Daniel B. Toso
  8. Abhay Kotecha
  9. Diana M. Bautista
  10. Stephen G. Brohawn

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

No abstract available

Article activity feed

  3. Version published to 10.1038/s41594-021-00619-0
  4. ScreenIT

    SciScore for 10.1101/2020.06.17.156554: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cloning and protein expression: The coding sequence for the 3a protein from SARS-Cov-2 was codon optimized for Spodoptera frugiperda (Sf9 cells) and synthesized (IDT, Newark, NJ).
    Sf9
    suggested: CLS Cat# 604328/p700_Sf9, RRID:CVCL_0549)
    Constructs (2 μg) were transfected into HEK293 cells on glass coverslips using Fugene HD (Promega, Madison, WI) per manufacturer’s instructions.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Software and Algorithms
    SentencesResources
    To calculate the total number of liposomes, we quantified the total number of liposomes using auto segmentation on MetaXpress 6 from brightfield images.
    MetaXpress
    suggested: (MetaXpress, RRID:SCR_016654)
    All statistical tests were performed using Prism (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Cryo-EM data processing: For the apo 3a dimer, motion-correction and dose-weighting were performed on all 6,309 movies using RELION 3.1’s implementation of MotionCor2, and 2x “binned” to 1.137 Å per pixel49–51.
    RELION
    suggested: (RELION, RRID:SCR_016274)
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    A set of 86,479 particles were identified, polished in RELION3.1 and refined in cryoSPARC v2 with subsequent homogeneous and non-uniform refinement54 jobs (maps were low-pass filtered to an initial resolution where TM density was still visible (6-9 Ă…), and the dynamic mask was tightened with the near (2-5 Ă…) and far (3-9 Ă…) parameters), yielding a map with overall resolution of 3.6 Ă….
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Molprobity61 was used to evaluate the stereochemistry and geometry of the structure for subsequent rounds of manual adjustment in Coot and refinement in Phenix.
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    Cavity measurements were made with HOLE64 implemented in Coot.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Figures were prepared using PyMOL, Chimera, ChimeraX66, Fiji, Prism, GNU Image Manipulation Program, and Adobe Photoshop and Illustrator software.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Image Manipulation Program
    suggested: (GNU Image Manipulation Program, RRID:SCR_003182)
    Adobe Photoshop
    suggested: (Adobe Photoshop, RRID:SCR_014199)
    Illustrator
    suggested: (Adobe Illustrator, RRID:SCR_010279)
    The samples were excited with 488nm argon laser and image analysis was performed using ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  5. Version published to 10.1101/2020.06.17.156554v3 on bioRxiv
  6. Version published to 10.1101/2020.06.17.156554v2 on bioRxiv
  7. ScreenIT

    SciScore for 10.1101/2020.06.17.156554: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    To test this , we generated an N-terminal deletion construct lacking the first 41 amino acids ( 3aΔN ) and compared its localization to wild-type 3a in HEK cells .
    HEK
    suggested: CLS Cat# 300192/p777_HEK293, CVCL_0045
    Protein purification For preparation of the 3a dimer and mutant constructs , infected Sf9 cells from 1 L of culture ( 15-20 mL of cell pellet ) were thawed in 100 mL of Lysis Buffer containing 50 mM Tris , 150 mM KCl , 1mM EDTA pH 8 .
    Sf9
    suggested: None
    Constructs (2 µg) were transfected into HEK293 cells on glass coverslips using Fugene HD (Promega, Madison, WI) per manufacturer’s instructions.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, CVCL_0045
    Software and Algorithms
    SentencesResources
    Cryo-EM data processing For the apo 3a dimer , motion-correction and dose-weighting were performed on all 6,309 movies using RELION 3.1’s implementation of MotionCor2 , and 2x “binned” to 1.137 Å per pixel30-32 .
    RELION
    suggested: (RELION, SCR_016274)
          <div style="margin-bottom:8px">
        <div><b>MotionCor2</b></div>
        <div>suggested: (MotionCor2, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016499">SCR_016499</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the tetramer, the highest resolution reconstruction came from cryoSPARC v2 non-uniform refinement with a tightened mask, which was subsequently used for dimer-docking and figure preparation.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>cryoSPARC</b></div>
        <div>suggested: (cryoSPARC, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016501">SCR_016501</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Molprobity39 was used to evaluate the stereochemistry and geometry of the structure for subsequent rounds of manual adjustment in Coot and refinement in Phenix.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Phenix</b></div>
        <div>suggested: (Phenix, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014224">SCR_014224</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cavity measurements were made with HOLE41 implemented in Coot.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Coot</b></div>
        <div>suggested: (Coot, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014222">SCR_014222</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Figures were prepared using PyMOL, Chimera, ChimeraX43 , Fiji, Prism, GNU Image Manipulation Program, and Adobe Photoshop and Illustrator software.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>PyMOL</b></div>
        <div>suggested: (PyMOL, <a href="https://scicrunch.org/resources/Any/search?q=SCR_000305">SCR_000305</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Fiji</b></div>
        <div>suggested: (Fiji, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002285">SCR_002285</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Image Manipulation Program</b></div>
        <div>suggested: (GNU Image Manipulation Program, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003182">SCR_003182</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Adobe Photoshop</b></div>
        <div>suggested: (Adobe Photoshop, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014199">SCR_014199</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Illustrator</b></div>
        <div>suggested: (Adobe Illustrator, <a href="https://scicrunch.org/resources/Any/search?q=SCR_010279">SCR_010279</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The samples were excited with 488nm argon laser and image analysis was performed using ImageJ.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>ImageJ</b></div>
        <div>suggested: (ImageJ, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003070">SCR_003070</a>)</div>
      </div>
    </td></tr></table>

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog).

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  8. Version published to 10.1101/2020.06.17.156554v1 on bioRxiv