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- PDB-6xm3: Structure of SARS-CoV-2 spike at pH 5.5, single RBD up, conformation 1 -

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Basic information

Entry
Database: PDB / ID: 6xm3
TitleStructure of SARS-CoV-2 spike at pH 5.5, single RBD up, conformation 1
ComponentsSpike glycoproteinPeplomer
KeywordsVIRAL PROTEIN / SARS-CoV-2 spike / COVID19
Function / homology
Function and homology information


suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / host cell surface receptor binding / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / suppression by virus of host type I interferon-mediated signaling pathway / viral entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope ...suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / host cell surface receptor binding / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / suppression by virus of host type I interferon-mediated signaling pathway / viral entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope / pathogenesis / host cell plasma membrane / virion membrane / integral component of membrane / identical protein binding
Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Spike glycoprotein, heptad repeat 2, coronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S1, C-terminal / Spike receptor binding domain superfamily, coronavirus / Betacoronavirus-like spike glycoprotein S1, N-terminal / Betacoronavirus spike glycoprotein S1, receptor binding
Spike glycoprotein / polysac:dglcpnacb1-4dglcpnacb1-:
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsZhou, T. / Tsybovsky, Y. / Olia, A. / Kwong, P.D.
CitationJournal: bioRxiv / Year: 2020
Title: A pH-dependent switch mediates conformational masking of SARS-CoV-2 spike.
Abstract: SARS-CoV-2 has emerged as a global pathogen, sparking urgent vaccine development efforts with the trimeric spike. However, the inability of antibodies like CR3022, which binds a cryptic spike epitope ...SARS-CoV-2 has emerged as a global pathogen, sparking urgent vaccine development efforts with the trimeric spike. However, the inability of antibodies like CR3022, which binds a cryptic spike epitope with nanomolar affinity, to neutralize virus, suggests a spike-based means of neutralization escape. Here, we show the SARS-CoV-2 spike to have 10% the unfolding enthalpy of a globular protein at physiological pH, where it is recognized by antibodies like CR3022, and up to 10-times more unfolding enthalpy at endosomal pH, where it sheds such antibodies, suggesting that the spike evades potentially neutralizing antibody through a pH-dependent mechanism of conformational masking. To understand the compatibility of this mechanism with ACE2-receptor interactions, we carried out binding measurements and determined cryo-EM structures of the spike recognizing up to three ACE2 molecules at both physiological and endosomal pH. In the absence of ACE2, cryo-EM analyses indicated lower pH to reduce conformational heterogeneity. Single-receptor binding domain (RBD)-up conformations dominated at pH 5.5, resolving into a locked all-down conformation at lower pH through lowering of RBD and refolding of a pH-dependent switch. Notably, the emerging Asp614Gly strain partially destabilizes the switch that locks RBD down, thereby enhancing functional interactions with ACE2 while reducing evasion by conformational masking.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 29, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 12, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Spike glycoprotein
B: Spike glycoprotein
C: Spike glycoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)435,82446
Polymers422,8583
Non-polymers12,96643
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 140952.531 Da / Num. of mol.: 3 / Mutation: R682G, R683S, R685S, K986P, V987P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Cell line (production host): Freestyle 293F / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#2: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 17
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#3: 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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 26 / 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
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: SARS-CoV-2 Spike / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.423 MDa / Experimental value: NO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Homo sapiens (human) / Cell: Freestyle 293F
Buffer solutionpH: 5.5
Buffer componentConc.: 0.1 M / Name: Acetate
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1250 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.18 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9816
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
2Latitudeimage acquisition
4CTFFIND4.1.13CTF correction
7UCSF Chimera1.14model fitting
8Coot0.8.9.2model fitting
10RELION3.0.8initial Euler assignment
11RELION3.0.8final Euler assignment
12RELION3.0.8classification
13RELION3.0.83D reconstruction
14PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2113090 / Details: Automated picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 247605 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 6XM0

6xm0

Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00725937
ELECTRON MICROSCOPYf_angle_d0.69335308
ELECTRON MICROSCOPYf_dihedral_angle_d12.1083724
ELECTRON MICROSCOPYf_chiral_restr0.0514198
ELECTRON MICROSCOPYf_plane_restr0.0054491

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