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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 dataSARS-CoV2 SPIKE protein reconstruction at 3.22 Angstrom
Sample
  • Virus: Severe acute respiratory syndrome coronavirus 2
    • Protein or peptide: Spike glycoproteinSpike protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
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 / host cell endoplasmic reticulum-Golgi intermediate compartment membrane ...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 / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / entry receptor-mediated virion attachment to host cell / receptor-mediated endocytosis of virus by host cell / Attachment and Entry / membrane fusion / 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 / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / membrane / identical protein binding / 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
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 ...Herrera 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.
History
DepositionMay 28, 2020-
Header (metadata) releaseJun 10, 2020-
Map releaseJun 10, 2020-
UpdateJan 27, 2021-
Current statusJan 27, 2021Processing 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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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22078.png

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)
AnnotationSARS-CoV2 SPIKE protein reconstruction at 3.22 Angstrom
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/NZ777686
NX/NY/NZ10710993
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
Components
  • Virus: Severe acute respiratory syndrome coronavirus 2
    • Protein or peptide: Spike glycoproteinSpike protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Severe acute respiratory syndrome coronavirus 2

SupramoleculeName: Severe acute respiratory syndrome coronavirus 2 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1 / NCBI-ID: 2697049
Sci species name: Severe acute respiratory syndrome coronavirus 2
Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: Yes / Virus empty: Yes
Host systemOrganism: Cricetulus griseus (Chinese hamster)

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Macromolecule #1: Spike glycoprotein

MacromoleculeName: Spike glycoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 140.824406 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: CVNLTTRTQL PPAYTNSFTR GVYYPDKVFR SSVLHSTQDL FLPFFSNVTW FHAIHVSGTN GTKRFDNPVL PFNDGVYFAS TEKSNIIRG WIFGTTLDSK TQSLLIVNNA TNVVIKVCEF QFCNDPFLGV YYHKNNKSWM ESEFRVYSSA NNCTFEYVSQ P FLMDLEGK ...String:
CVNLTTRTQL PPAYTNSFTR GVYYPDKVFR SSVLHSTQDL FLPFFSNVTW FHAIHVSGTN GTKRFDNPVL PFNDGVYFAS TEKSNIIRG WIFGTTLDSK TQSLLIVNNA TNVVIKVCEF QFCNDPFLGV YYHKNNKSWM ESEFRVYSSA NNCTFEYVSQ P FLMDLEGK QGNFKNLREF VFKNIDGYFK IYSKHTPINL VRDLPQGFSA LEPLVDLPIG INITRFQTLL ALHRSYLTPG DS SSGWTAG AAAYYVGYLQ PRTFLLKYNE NGTITDAVDC ALDPLSETKC TLKSFTVEKG IYQTSNFRVQ PTESIVRFPN ITN LCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQ TGKIA DYNYKLPDDF TGCVIAWNSN NLDSKVGGNY NYLYRLFRKS NLKPFERDIS TEIYQAGSTP CNGVEGFNCY FPLQS YGFQ PTNGVGYQPY RVVVLSFELL HAPATVCGPK KSTNLVKNKC VNFNFNGLTG TGVLTESNKK FLPFQQFGRD IADTTD AVR DPQTLEILDI TPCSFGGVSV ITPGTNTSNQ VAVLYQDVNC TEVPVAIHAD QLTPTWRVYS TGSNVFQTRA GCLIGAE HV NNSYECDIPI GAGICASYQT QTNSPGSASS VASQSIIAYT MSLGAENSVA YSNNSIAIPT NFTISVTTEI LPVSMTKT S VDCTMYICGD STECSNLLLQ YGSFCTQLNR ALTGIAVEQD KNTQEVFAQV KQIYKTPPIK DFGGFNFSQI LPDPSKPSK RSFIEDLLFN KVTLADAGFI KQYGDCLGDI AARDLICAQK FNGLTVLPPL LTDEMIAQYT SALLAGTITS GWTFGAGAAL QIPFAMQMA YRFNGIGVTQ NVLYENQKLI ANQFNSAIGK IQDSLSSTAS ALGKLQDVVN QNAQALNTLV KQLSSNFGAI S SVLNDILS RLDPPEAEVQ IDRLITGRLQ SLQTYVTQQL IRAAEIRASA NLAATKMSEC VLGQSKRVDF CGKGYHLMSF PQ SAPHGVV FLHVTYVPAQ EKNFTTAPAI CHDGKAHFPR EGVFVSNGTH WFVTQRNFYE PQIITTDNTF VSGNCDVVIG IVN NTVYDP LQPELDSFKE ELDKYFKNHT SPDVDLGDIS GINASVVNIQ KEIDRLNEVA KNLNESLIDL QELGKYEQGS GYIP EAPRD GQAYVRKDGE WVLLSTFLGR SLEVLFQGPG HHHHHHHHSA WSHPQFEKGG GSGGGGSGGS AWSHPQFEK

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 33 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
250.0 mMNaClSodium chloridesodium chloride
50.0 mMTris
GridModel: UltrAuFoil / Material: GOLD / Mesh: 400 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 1131 / Average exposure time: 2.5 sec. / Average electron dose: 66.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 75582
CTF correctionSoftware - Name: cryoSPARC (ver. 2.14.2)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.14.2)
Final 3D classificationNumber classes: 76 / Software - Name: cryoSPARC (ver. 2.14.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.14.2)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14.2) / Number images used: 54395
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6vxx


Chain - Chain ID: A / Chain - Residue range: 27-1147
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 120 / Target criteria: CC
Output model

PDB-6x6p:
Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

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