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- EMDB-22078: Characterization of the SARS-CoV-2 S Protein: Biophysical, Bioche... -

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Basic information

Entry
Database: EMDB / ID: EMD-22078
TitleCharacterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis
Map data
SampleSevere acute respiratory syndrome coronavirus 2:
virus / SARS-CoV-2 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: 3.22 Å
AuthorsHerrera NG / Morano NC / Celikgil A / Georgiev GI / Malonis R / Lee JH / Tong K / Vergnolle O / Massimi A / Yen LY / Noble AJ / Kopylov M / Bonanno JB / Garrett-Thompson SC / Hayes DB / Brenowitz M / Garforth SJ / Eng ET / Lai JR / Almo SC
CitationJournal: bioRxiv / Year: 2020
Title: Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.
Authors: Natalia G Herrera / Nicholas C Morano / Alev Celikgil / George I Georgiev / Ryan J Malonis / James H Lee / Karen Tong / Olivia Vergnolle / Aldo B Massimi / Laura Y Yen / Alex J Noble / ...Authors: Natalia G Herrera / Nicholas C Morano / Alev Celikgil / George I Georgiev / Ryan J Malonis / James H Lee / Karen Tong / Olivia Vergnolle / Aldo B Massimi / Laura Y Yen / Alex J Noble / Mykhailo Kopylov / Jeffrey B Bonanno / Sarah C Garrett-Thomson / David B Hayes / Robert H Bortz / Ariel S Wirchnianski / Catalina Florez / Ethan Laudermilch / Denise Haslwanter / J Maximilian Fels / M Eugenia Dieterle / Rohit K Jangra / Jason Barnhill / Amanda Mengotto / Duncan Kimmel / Johanna P Daily / Liise-Anne Pirofski / Kartik Chandran / Michael Brenowitz / Scott J Garforth / Edward T Eng / Jonathan R Lai / Steven C Almo
Abstract: Coronavirus disease 2019 ( ) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 ( ), and there is a critical need to produce large quantities of high- ...Coronavirus disease 2019 ( ) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 ( ), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike ( ) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural ( ) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.
Validation ReportPDB-ID: 6x6p

SummaryFull reportAbout validation report
History
DepositionMay 28, 2020-
Header (metadata) releaseJun 10, 2020-
Map releaseJun 10, 2020-
UpdateJun 10, 2020-
Current statusJun 10, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6x6p
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_22078.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 384 pix.
= 406.272 Å
1.06 Å/pix.
x 384 pix.
= 406.272 Å
1.06 Å/pix.
x 384 pix.
= 406.272 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.058 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.47792593 - 1.1256504
Average (Standard dev.)0.0005189992 (±0.020042568)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 406.27197 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0581.0581.058
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z406.272406.272406.272
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.4781.1260.001

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Supplemental data

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Sample components

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Entire Severe acute respiratory syndrome coronavirus 2

EntireName: Severe acute respiratory syndrome coronavirus 2 / Number of components: 3

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Component #1: virus, Severe acute respiratory syndrome coronavirus 2

VirusName: Severe acute respiratory syndrome coronavirus 2 / Class: VIRION / Empty: Yes / Enveloped: Yes / Isolate: OTHER
SpeciesSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Cricetulus griseus (Chinese hamster)

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Component #2: protein, SARS-CoV-2 spike glycoprotein

ProteinName: SARS-CoV-2 spike glycoprotein / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 140.824406 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Cricetulus griseus (Chinese hamster)

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Component #3: ligand, N-ACETYL-D-GLUCOSAMINE

LigandName: N-ACETYL-D-GLUCOSAMINEN-Acetylglucosamine / Number of Copies: 57 / 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.: 1 mg/mL / pH: 8
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 %

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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: 66.5 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD / Defocus: 800.0 - 2500.0 nm
Specimen HolderModel: OTHER
CameraDetector: OTHER

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Image acquisition

Image acquisitionNumber of digital images: 1131

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C3 (3 fold cyclic) / Number of projections: 54395
3D reconstructionSoftware: cryoSPARC / Resolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Target criteria: CC / Refinement space: REAL
Input PDB model: 6VXX
Chain ID: A

Overall bvalue: 120
Output model

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