Ensemble cryo-electron microscopy reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication-transcription complex

  1. James Chen
  2. Qi Wang
  3. Brandon Malone
  4. Eliza Llewellyn
  5. Yakov Pechersky
  6. Kashyap Maruthi
  7. Ed T. Eng
  8. Jason K. Perry
  9. Elizabeth A. Campbell
  10. David E. Shaw
  11. Seth A. Darst

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Abstract

The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex, or RTC) associated with two copies of nsp13 (nsp13 2 -RTC). One copy of nsp13 interacts with the template RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backwards on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here, we use cryo-electron microscopy and molecular dynamics simulations to analyze the nsp13 2 -RTC, revealing four distinct conformational states of the helicases. The results suggest a mechanism for the nsp13 2 -RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site.

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    SciScore for 10.1101/2021.11.10.468168: (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

    Recombinant DNA
    SentencesResources
    Briefly, a pRSFDuet-1 plasmid containing His6-SUMO SARS-CoV-2 nsp12 (Addgene #159107) was transformed into E. coli BL21-CodonPlus cells (Agilent).
    pRSFDuet-1
    suggested: RRID:Addgene_165451)
    Briefly, the pCDFDuet-1 plasmid containing His6 SARS-CoV-2 nsp7/8 (Addgene #159092) was transformed into E. coli BL21 (DE3).
    pCDFDuet-1
    suggested: RRID:Addgene_15917)
    Briefly, the pet28 plasmid containing His6 SARS-CoV-2 nsp13 (Addgene #159390) was transformed into E. coli Rosetta (DE3) (Novagen).
    pet28
    suggested: RRID:Addgene_21766)
    Software and Algorithms
    SentencesResources
    Flow-through was collected, concentrated by centrifugal filtration (Amicon), and loaded on a Superdex 200 Hiload 16/600 (Cytiva).
    Amicon
    suggested: None
    Dose-fractionated movies were gain-normalized, drift-corrected, summed, and dose-weighted using MotionCor2 46.
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    Particles were sorted using two rounds of cryoSPARC 2D classification (N=100, where N equals the number of classes), resulting in 661,105 curated particles that were re-extracted with a boxsize of 320 px.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Locally refined maps were combined into an nsp132-RTC composite map using PHENIX ‘Combine Focused Maps’ 50,51, with resulting nominal resolution of 2.8 Å.
    PHENIX
    suggested: (Phenix, RRID:SCR_014224)
    S1), followed by masked RELION 3D classification.
    RELION
    suggested: (RELION, RRID:SCR_016274)
    Local resolution calculations were generated using blocres and blocfilt from the Bsoft package 29.
    Bsoft
    suggested: (Bsoft, RRID:SCR_016503)
    Models were inspected and modified in Coot 53.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Simulation structures shown in figures were rendered using PyMol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).
    PyMol
    suggested: (PyMOL, RRID:SCR_000305)
    The quantification and statistical analyses for model refinement and validation were generated using MolProbity 65 and PHENIX 51.
    MolProbity
    suggested: (MolProbity, RRID:SCR_014226)
    The cryo-EM density maps and atomic coordinates have been deposited in the EMDataBank and Protein Data Bank as follows: nsp131-RTC (EMD-24431, 7RE2), nsp132-RTC (composite) (EMD-24430, 7RE1), (nsp132-RTC)2 (EMD-24432, 7RE3), nsp132-RTC (nsp13.1-apo) (EMD-24428, 7RDZ), nsp132-RTC (nsp13.1-engaged) (EMD-24427, 7RDY), nsp132-RTC (nsp13.1-swiveled) (EMD-24429, 7RE0), nsp132-RTC (1B-open) (EMD-24426, 7RDX).
    EMDataBank
    suggested: (EMDataResource.org, RRID:SCR_003207)

    Results from OddPub: Thank you for sharing your data.

    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.

    Results from scite Reference Check: We found no unreliable references.

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  2. Version published to 10.1101/2021.11.10.468168v1 on bioRxiv