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- EMDB-11496: Subtomogram averaging of SARS-CoV-2 spike protein from unconcentr... -

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Basic information

Entry
Database: EMDB / ID: EMD-11496
TitleSubtomogram averaging of SARS-CoV-2 spike protein from unconcentrated virions: Prefusion Class (2 open RBDs)
Map dataprefusion 2 open RBDs
Sample
  • Virus: Severe acute respiratory syndrome coronavirus 2
    • Protein or peptide: SARS-CoV-2 Spike proteins on virions
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 / membrane fusion / Attachment and Entry / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / host cell surface receptor binding / symbiont-mediated suppression of host innate immune response / 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 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, betacoronavirus / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, betacoronavirus / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsubtomogram averaging / cryo EM / Resolution: 9.9 Å
AuthorsKe Z / Oton J / Zivanov J / Lu JM / Peukes J / Cortese M / Zila V / Scheres SHW / Briggs JAG
Funding supportEuropean Union, United Kingdom, Japan, Germany, 5 items
OrganizationGrant numberCountry
European Research Council (ERC)ERC-CoG-648432European Union
Medical Research Council (MRC, United Kingdom)MC_UP_A025_1013 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/16 United Kingdom
Japan Society for the Promotion of Science (JSPS) Japan
German Research Foundation (DFG)240245660-SFB 1129 Germany
CitationJournal: Nature / Year: 2020
Title: Structures and distributions of SARS-CoV-2 spike proteins on intact virions.
Authors: Zunlong Ke / Joaquin Oton / Kun Qu / Mirko Cortese / Vojtech Zila / Lesley McKeane / Takanori Nakane / Jasenko Zivanov / Christopher J Neufeldt / Berati Cerikan / John M Lu / Julia Peukes / ...Authors: Zunlong Ke / Joaquin Oton / Kun Qu / Mirko Cortese / Vojtech Zila / Lesley McKeane / Takanori Nakane / Jasenko Zivanov / Christopher J Neufeldt / Berati Cerikan / John M Lu / Julia Peukes / Xiaoli Xiong / Hans-Georg Kräusslich / Sjors H W Scheres / Ralf Bartenschlager / John A G Briggs /
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind to the ...Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.
History
DepositionJul 28, 2020-
Header (metadata) releaseAug 5, 2020-
Map releaseAug 5, 2020-
UpdateDec 30, 2020-
Current statusDec 30, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11496.png

Downloads & links

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Map

FileDownload / File: emd_11496.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationprefusion 2 open RBDs
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.06 Å/pix.
x 128 pix.
= 392.192 Å		3.06 Å/pix.
x 128 pix.
= 392.192 Å		3.06 Å/pix.
x 128 pix.
= 392.192 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.064 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.4 / Movie #1: 3.4
Minimum - Maximum-3.9059904 - 15.108139
Average (Standard dev.)-0.025958644 (±0.8509294)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 392.192 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.0643.0643.064
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z392.192392.192392.192
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-3.90615.108-0.026

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Supplemental data

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Half map: prefusion 2 open RBDs, half1

Fileemd_11496_half_map_1.map
Annotationprefusion 2 open RBDs, half1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: prefusion 2 open RBDs, half2

Fileemd_11496_half_map_2.map
Annotationprefusion 2 open RBDs, half2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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: SARS-CoV-2 Spike proteins on virions

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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: all
Details: Unconcentrated supernatant of SARS-CoV-2 virions produced from infected VeroE6 cells.
NCBI-ID: 2697049
Sci species name: Severe acute respiratory syndrome coronavirus 2
Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No

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Macromolecule #1: SARS-CoV-2 Spike proteins on virions

MacromoleculeName: SARS-CoV-2 Spike proteins on virions / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2 / Strain: Germany/BavPat1/2020
SequenceString: MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY ...String:
MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY VSQPFLMDLE GKQGNFKNLR EFVFKNIDGY FKIYSKHTPI NLVRDLPQGF SALEPLVDLP IGINITRFQT LLALHRSYLT PGDSSSGWTA GAAAYYVGYL QPRTFLLKYN ENGTITDAVD CALDPLSETK CTLKSFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV VVLSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG VLTESNKKFL PFQQFGRDIA DTTDAVRDPQ TLEILDITPC SFGGVSVITP GTNTSNQVAV LYQGVNCTEV PVAIHADQLT PTWRVYSTGS NVFQTRAGCL IGAEHVNNSY ECDIPIGAGI CASYQTQTNS PRRARSVASQ SIIAYTMSLG AENSVAYSNN SIAIPTNFTI SVTTEILPVS MTKTSVDCTM YICGDSTECS NLLLQYGSFC TQLNRALTGI AVEQDKNTQE VFAQVKQIYK TPPIKDFGGF NFSQILPDPS KPSKRSFIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFGA GAALQIPFAM QMAYRFNGIG VTQNVLYENQ KLIANQFNSA IGKIQDSLSS TASALGKLQD VVNQNAQALN TLVKQLSSNF GAISSVLNDI LSRLDKVEAE VQIDRLITGR LQSLQTYVTQ QLIRAAEIRA SANLAATKMS ECVLGQSKRV DFCGKGYHLM SFPQSAPHGV VFLHVTYVPA QEKNFTTAPA ICHDGKAHFP REGVFVSNGT HWFVTQRNFY EPQIITTDNT FVSGNCDVVI GIVNNTVYDP LQPELDSFKE E(NAG)(NAG)(NAG)(NAG)(NAG)(NAG)(NAG)(NAG)(NAG) (NAG)(NAG)(NAG)(NAG)(NAG)(NAG)(NAG)(NAG)(NAG)(NAG) (NAG)(NAG)LDKYFKNH TSPDVDLGDI SGINASVVNI QKEIDRLNEV AKNLNESLID LQELGKYEQY IKWPWYIWLG FIAGLIAIVM VTIMLCCMTS CCSCLKGCCS CGSCCKFDED DSEPVLKGVK LHYT

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
GridModel: C-flat-2/2 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-10 / Average exposure time: 1.25 sec. / Average electron dose: 3.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 9.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0) / Software - details: subtomogram averaging
Details: Modified version of RELION for subtomogram averaging
Number subtomograms used: 525
ExtractionNumber tomograms: 156 / Number images used: 4104 / Software: (Name: MATLAB (ver. 2016b), RELION (ver. 3.1.0)) / Details: Matlab Relion dedicated python scripts
CTF correctionSoftware: (Name: NOVACTF (ver. 1.0.0), IMOD (ver. 4.10.30), RELION (ver. 3.1.0))
Details: novaCTF Relion dedicated python script
Final 3D classificationSoftware - Name: RELION (ver. 3.1.0) / Software - details: subtomogram averaging
Details: Focused classification without alignment and with partial signal subtraction of the RBD region. Selection of 2 open RBDs spikes.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.0) / Software - details: subtomogram averaging
Details: Modified version of RELION for subtomogram averaging

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