Distinct conformational states of SARS-CoV-2 spike protein
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Abstract
The ongoing SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic has created urgent needs for intervention strategies to control the crisis. The spike (S) protein of the virus forms a trimer and catalyzes fusion between viral and target cell membranes - the first key step of viral infection. Here we report two cryo-EM structures, both derived from a single preparation of the full-length S protein, representing the prefusion (3.1Å resolution) and postfusion (3.3Å resolution) conformations, respectively. The spontaneous structural transition to the postfusion state under mild conditions is independent of target cells. The prefusion trimer forms a tightly packed structure with three receptor-binding domains clamped down by a segment adjacent to the fusion peptide, significantly different from recently published structures of a stabilized S ectodomain trimer. The postfusion conformation is a rigid tower-like trimer, but decorated by N-linked glycans along its long axis with almost even spacing, suggesting possible involvement in a mechanism protecting the virus from host immune responses and harsh external conditions. These findings advance our understanding of how SARS-CoV-2 enters a host cell and may guide development of vaccines and therapeutics.
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SciScore for 10.1101/2020.05.16.099317: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Western blot: Western blot was performed either using an anti-strep tag antibody or anti-SARA-COV-2 S antibody following a protocol described previously50. anti-SARA-COV-2 Ssuggested: NoneMembranes were blocked with 5% skimmed milk in PBS for 1 hour and incubated either with anti-strep tag antibody (IBA Lifesciences) or anti-SARS-COV-2 polyclone antibody (Sino Biological Inc.) for another hour at room temperature. anti-strep tagsuggested: Noneanti-SARS-COV-2SciScore for 10.1101/2020.05.16.099317: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Western blot: Western blot was performed either using an anti-strep tag antibody or anti-SARA-COV-2 S antibody following a protocol described previously50. anti-SARA-COV-2 Ssuggested: NoneMembranes were blocked with 5% skimmed milk in PBS for 1 hour and incubated either with anti-strep tag antibody (IBA Lifesciences) or anti-SARS-COV-2 polyclone antibody (Sino Biological Inc.) for another hour at room temperature. anti-strep tagsuggested: Noneanti-SARS-COV-2suggested: NoneAlkaline phosphatase conjugated anti-Rabbit IgG (1:5000) (Sigma) was used as a secondary antibody. anti-Rabbit IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Varying amount of the full-length SARS-CoV2 S protein expression construct (0.1-10 µg) and the α fragment of β-galactosidase construct (10 µg), or the full-length ACE2 expression construct (10 µg) together with the ω fragment of β-galactosidase construct (10 µg), were mixed with DMEM medium (Gibco) containing PEI (80 µg) and added to HEK293T cells. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Software and Algorithms Sentences Resources Automated data collection was carried out using SerialEM version65 at a magnification of 105,000× and the K3 detector in counting mode (pixel size, 0.825 Å) at a dose rate of ∼1.1 electrons per physical pixels per second. SerialEMsuggested: (SerialEM, RRID:SCR_017293)For the S1 fragment data set, CrYOLO53 was used for particle picking, RELION 3.0.8 was used for 2D classification, 3D classification and refinement. RELIONsuggested: (RELION, RRID:SCR_016274)Structural biology applications used in this project were compiled and configured by SBGrid56 Model building: The initial templates for model building used the stabilized SARS-CoV-2 S ectodomain trimer structure (PDB ID 6vxx) for the prefusion conformation, and a homology model, calculated by I-TASSER, of S2 postfusion trimer structure from mouse hepatitis virus (MHV) (PDB ID: 6b3o) for the postfusion confirmation. I-TASSERsuggested: (I-TASSER, RRID:SCR_014627)Several rounds of manual building were performed in Coot. Cootsuggested: (Coot, RRID:SCR_014222)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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 26 and 23. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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.
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