Cryo-EM Structures of the N501Y SARS-CoV-2 Spike Protein in Complex with ACE2 and Two Potent Neutralizing Antibodies
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Abstract
The recently reported “UK variant” of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-EM structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. The additional interactions result in increased affinity of ACE2 for the N501Y mutant, accounting for its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to two representative potent neutralizing antibody fragments.
Short summary
The N501Y mutation found in the coronavirus UK variant increases infectivity but some neutralizing antibodies can still bind.
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SciScore for 10.1101/2021.01.11.426269: (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 After washing, wells were incubated with either goat anti-human IgG (Jackson ImmunoResearch) or goat anti-Mouse IgG Fc Secondary Antibody, HRP (Invitrogen) at a 1:8,000 dilution in PBS-T + 0.5% BSA buffer for 1 h at room temperature. anti-human IgGsuggested: NoneAfter washing, wells were incubated at a 1:8,000 dilution of Goat anti-Mouse IgG Fc Secondary Antibody, HRP (Invitrogen) in PBS-T + 0.5% BSA buffer for 1 h at room temperature. anti-Mouse IgGsuggeste…SciScore for 10.1101/2021.01.11.426269: (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 After washing, wells were incubated with either goat anti-human IgG (Jackson ImmunoResearch) or goat anti-Mouse IgG Fc Secondary Antibody, HRP (Invitrogen) at a 1:8,000 dilution in PBS-T + 0.5% BSA buffer for 1 h at room temperature. anti-human IgGsuggested: NoneAfter washing, wells were incubated at a 1:8,000 dilution of Goat anti-Mouse IgG Fc Secondary Antibody, HRP (Invitrogen) in PBS-T + 0.5% BSA buffer for 1 h at room temperature. anti-Mouse IgGsuggested: NoneFor all experiments, controls for antibody–BSA interactions were performed. antibody–BSAsuggested: NoneExperimental Models: Cell Lines Sentences Resources SARS-CoV-2 S and SARS_CoV-2 S N501Y pseudotyped retroviral particles were produced in HEK293T cells as described previously (29). HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)For cell-entry and neutralization assays, HEK293T-ACE2 cells were seeded in 96-well plates at 50,000 cells per well. HEK293T-ACE2suggested: NoneSoftware and Algorithms Sentences Resources For negative stain data, motion correction and CTF estimation were performed in RELION v. RELIONsuggested: (RELION, RRID:SCR_016274)For cryo-EM data, motion correction in patch mode (EER upsampling factor 1, EER number of fractions 40), CTF estimation in patch mode, reference-free particle picking, and particle extraction (extraction box size 640, Fourier crop to box size 320) were performed on-the-fly in cryoSPARC. cryoSPARCsuggested: (cryoSPARC, RRID:SCR_016501)Initial models were first refined against sharpened locally refined maps, followed by iterative rounds of refinement against consensus map in COOT v. COOTsuggested: (Coot, RRID:SCR_014222)Model validation was performed using MolProbity (33). MolProbitysuggested: (MolProbity, RRID:SCR_014226)1.1.1 (34), and PyMOL (v.2.2 Schrodinger, LLC) PyMOLsuggested: (PyMOL, RRID:SCR_000305)The IC50 values were calculated using a four-parameter dose-response (sigmoidal) curve in GraphPad Prism (version 9 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com). GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)The density maps reported will be made publicly available in the Electron Microscopy Data Bank (EMDB: https://www.ebi.ac.uk/pdbe/emdb/) and atomic coordinates for the structures will be available at the Protein Data Bank (PDB: https://www.rcsb.org). https://www.ebi.ac.uk/pdbe/emdb/suggested: (Electron Microscopy Data Bank at PDBe (MSD-EBI, RRID:SCR_006506)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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 32, 27, 29 and 31. 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.
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- No protocol registration statement was detected.
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