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- PDB-7zbu: CryoEM structure of SARS-CoV-2 spike monomer in complex with neut... -

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Basic information

Entry
Database: PDB / ID: 7zbu
TitleCryoEM structure of SARS-CoV-2 spike monomer in complex with neutralising antibody P008_60
Components
  • P008_60 antibody, Heavy chain
  • P008_60 antibody, Light chain
  • Spike glycoprotein
KeywordsVIRAL PROTEIN / covid-19 / SARS-CoV-2 / spike / neutralizing antibody / immunity
Function / homology
Function and homology information


Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell ...Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated endocytosis of virus by host cell / Attachment and Entry / membrane fusion / symbiont-mediated suppression of host innate immune response / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / identical protein binding / membrane / plasma membrane
Similarity search - Function
Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal
Similarity search - Domain/homology
Chem-3Q9 / Spike glycoprotein
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.31 Å
AuthorsRosa, A. / Pye, V.E. / Cronin, N. / Cherepanov, P.
Funding support United Kingdom, 5items
OrganizationGrant numberCountry
The Francis Crick InstituteFC001061 United Kingdom
Wellcome TrustFC001061 United Kingdom
Medical Research Council (MRC, United Kingdom)FC001061 United Kingdom
Cancer Research UKFC001061 United Kingdom
Wellcome Trust206175/Z/17/Z United Kingdom
CitationJournal: Cell Rep / Year: 2022
Title: A neutralizing epitope on the SD1 domain of SARS-CoV-2 spike targeted following infection and vaccination.
Authors: Jeffrey Seow / Hataf Khan / Annachiara Rosa / Valeria Calvaresi / Carl Graham / Suzanne Pickering / Valerie E Pye / Nora B Cronin / Isabella Huettner / Michael H Malim / Argyris Politis / ...Authors: Jeffrey Seow / Hataf Khan / Annachiara Rosa / Valeria Calvaresi / Carl Graham / Suzanne Pickering / Valerie E Pye / Nora B Cronin / Isabella Huettner / Michael H Malim / Argyris Politis / Peter Cherepanov / Katie J Doores /
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the target for neutralizing antibodies elicited following both infection and vaccination. While extensive research has shown that ...Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the target for neutralizing antibodies elicited following both infection and vaccination. While extensive research has shown that the receptor binding domain (RBD) and, to a lesser extent, the N-terminal domain (NTD) are the predominant targets for neutralizing antibodies, identification of neutralizing epitopes beyond these regions is important for informing vaccine development and understanding antibody-mediated immune escape. Here, we identify a class of broadly neutralizing antibodies that bind an epitope on the spike subdomain 1 (SD1) and that have arisen from infection or vaccination. Using cryo-electron microscopy (cryo-EM) and hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), we show that SD1-specific antibody P008_60 binds an epitope that is not accessible within the canonical prefusion states of the SARS-CoV-2 spike, suggesting a transient conformation of the viral glycoprotein that is vulnerable to neutralization.
History
DepositionMar 24, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 17, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 24, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_DOI
Revision 1.2Aug 31, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Spike glycoprotein
H: P008_60 antibody, Heavy chain
L: P008_60 antibody, Light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)194,17112
Polymers191,8133
Non-polymers2,3589
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7120 Å2
ΔGint-7 kcal/mol
Surface area53670 Å2

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Components

#1: Protein Spike glycoprotein / S glycoprotein / E2 / Peplomer protein


Mass: 142269.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#2: Antibody P008_60 antibody, Heavy chain


Mass: 25954.072 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human) / Strain (production host): FreeStyle293
#3: Antibody P008_60 antibody, Light chain


Mass: 23589.068 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human) / Strain (production host): FreeStyle293
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Chemical ChemComp-3Q9 / 3-[5-[(4-ethyl-3-methyl-5-oxidanylidene-pyrrol-2-yl)methyl]-2-[[5-[(3-ethyl-4-methyl-5-oxidanylidene-pyrrol-2-yl)methyl]-3-(3-hydroxy-3-oxopropyl)-4-methyl-1H-pyrrol-2-yl]methyl]-4-methyl-1H-pyrrol-3-yl]propanoic acid


Mass: 588.694 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C33H40N4O6
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Monomeric Spike glycoprotein ectodomain from SARS-CoV-2 in complex with a neutralising antibody P008_60
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.125 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Details: 0.1% n-octyl glucoside in 150 mM NaCl, 20 mM Tris-HCl, pH8.0
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: SARS-CoV-2 spike ectodomain (0.5 mg/ml), supplemented 0.2 mg/ml P008_60 Fab and 0.1% n-octyl glucoside in 150 mM NaCl, 20 mM Tris-HCl, pH8.0
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Details: blotted for 3 to 4 sec before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3600 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.1 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 16624
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: dev_4213: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatch0.53particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
7UCSF Chimeramodel fitting
11RELION3.1classification
12cryoSPARC23D reconstruction
13PHENIX4213model refinement
Image processingDetails: The micrograph stacks were aligned, binned to the physical pixel size of 1.1 A and summed, with dose weighting applied, as implemented in MotionCor2
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1740430
Details: Initially, particles were picked with Gautomatch-v0.53 using 2D class averages of the trimeric spike, low-pass filtered to 20 A resolution, as templates. The resulting 1,740,430 particles, ...Details: Initially, particles were picked with Gautomatch-v0.53 using 2D class averages of the trimeric spike, low-pass filtered to 20 A resolution, as templates. The resulting 1,740,430 particles, extracted in Relion-3.1 and binned to a pixel size of 4.4 A, were subjected to two rounds of reference-free 2D classification in cryoSPARC-2. 283,956 particles belonging to well-defined 2D classes were subjected to classification into twelve 3D classes in Relion-3.1. Neither the 2D nor 3D class averages of the trimeric spike revealed features attributable to a bound Fab molecule. Next, 3,772,722 particles were picked using 2D class averages of the dissociated spikes. Following two rounds of 2D classification in cryoSPARC-2, 753,837 particles, re-extracted with pixel size of 2.2 A, were subjected to 3D classification in Relion-3.1 into 9 classes using an initial model obtained by Ab-initio reconstruction in cryoSPARC-2. The procedure revealed a single well-defined 3D class containing 208,343 particles (27.4%) of S1 protein with a bound Fab molecule. The particles, re-extracted without binning (with a pixel size 1.1 A), were subjected to two rounds of 3D classification using Ab-initio reconstruction in cryoSPARC-2 with 2 classes and class similarity set to 0. At the end of each round, the most populated class was selected, resulting in the final set of 166,619 particles.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.31 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 166619 / Algorithm: FOURIER SPACE / Details: Non Uniform refinement in cryoSPARC / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 81 / Protocol: OTHER / Space: REAL / Target criteria: Correlation coefficient
Details: The atomistic models of monomeric SARS-CoV-2 S1 protein and a Fab molecule, extracted from PDB entries 7A92 and 5WI9, were docked in the cryo-EM map using Chimera. The NTD and the RBD of the ...Details: The atomistic models of monomeric SARS-CoV-2 S1 protein and a Fab molecule, extracted from PDB entries 7A92 and 5WI9, were docked in the cryo-EM map using Chimera. The NTD and the RBD of the spike subunit were replaced with the crystal structures from PDB entries 7B62 and 7OAO, respectively. Guided by the cryo-EM map, the model was adjusted and extended interactively in Coot and refined using phenix.real_space_refine.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
17A92

7a92

A1
25WI9

5wi9

E1
35WI9

5wi9

F1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038740
ELECTRON MICROSCOPYf_angle_d0.6611897
ELECTRON MICROSCOPYf_dihedral_angle_d5.3941258
ELECTRON MICROSCOPYf_chiral_restr0.0521337
ELECTRON MICROSCOPYf_plane_restr0.0051550

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