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- EMDB-22256: Structure of SARS-CoV-2 spike at pH 5.5, all RBDs down -

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Basic information

Entry
Database: EMDB / ID: EMD-22256
TitleStructure of SARS-CoV-2 spike at pH 5.5, all RBDs down
Map data
SampleSARS-CoV-2 Spike:
Spike glycoprotein / ligand
Function / homology
Function and homology information


host cell endoplasmic reticulum-Golgi intermediate compartment membrane / host cell surface receptor binding / viral entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / pathogenesis / host cell plasma membrane / virion membrane / integral component of membrane
Spike glycoprotein S2, coronavirus / Spike receptor binding domain, betacoronavirus / Spike glycoprotein, heptad repeat 2, coronavirus / Spike receptor binding domain superfamily, coronavirus
Spike glycoprotein
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsTsybovsky Y / Zhou T / Olia A / Kwong PD
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 ReportPDB-ID: 6xm5

SummaryFull reportAbout validation report
History
DepositionJun 29, 2020-
Header (metadata) releaseJul 29, 2020-
Map releaseJul 29, 2020-
UpdateAug 12, 2020-
Current statusAug 12, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xm5
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_22256.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 440 pix.
= 374. Å
0.85 Å/pix.
x 440 pix.
= 374. Å
0.85 Å/pix.
x 440 pix.
= 374. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.012 / Movie #1: 0.012
Minimum - Maximum-0.04197237 - 0.0791549
Average (Standard dev.)0.0000783408 (±0.0013103698)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 374.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.850.850.85
M x/y/z440440440
origin x/y/z0.0000.0000.000
length x/y/z374.000374.000374.000
α/β/γ90.00090.00090.000
start NX/NY/NZ434333
NX/NY/NZ116118137
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS440440440
D min/max/mean-0.0420.0790.000

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Supplemental data

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Segmentation: #1

Fileemd_22256_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Refined map before post-processing

Fileemd_22256_additional.map
AnnotationRefined map before post-processing
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half-map 1

Fileemd_22256_half_map_1.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half-map 2

Fileemd_22256_half_map_2.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire SARS-CoV-2 Spike

EntireName: SARS-CoV-2 Spike / Number of components: 3

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Component #1: protein, SARS-CoV-2 Spike

ProteinName: SARS-CoV-2 Spike / Recombinant expression: No
MassTheoretical: 423 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: Freestyle 293F

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Component #2: protein, Spike glycoprotein

ProteinName: Spike glycoprotein / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 140.952531 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Homo sapiens (human)

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Component #3: ligand, 2-acetamido-2-deoxy-beta-D-glucopyranose

LigandName: 2-acetamido-2-deoxy-beta-D-glucopyranose / Number of Copies: 26 / Recombinant expression: No
MassTheoretical: 0.221208 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.5 mg/mL / pH: 5.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 95 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 105000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1250.0 - 2500.0 nm / Energy filter: GIF Bioquantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: OTHER

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Image acquisition

Image acquisitionNumber of digital images: 9816 / Sampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 108053
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Input PDB model: 6XM0
Output model

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