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- EMDB-22934: Negative stain electron microscopy reconstruction of rS2d-hexapro... -

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Basic information

Entry
Database: EMDB / ID: EMD-22934
TitleNegative stain electron microscopy reconstruction of rS2d-hexapro SARS-CoV-2 spike ectodomain
Map dataUnsharpened map of rS2d-hexapro SARS-CoV-2 spike ectodomain
Sample
  • Complex: SARS-CoV-2 rS2d Down State Spike Protein Trimer
    • Protein or peptide: rS2d-hexapro SARS-CoV-2 spike ectodomain
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / negative staining / Resolution: 10.7 Å
AuthorsEdwards RJ / Mansouri K
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI145687 United States
CitationJournal: bioRxiv / Year: 2020
Title: Cold sensitivity of the SARS-CoV-2 spike ectodomain.
Authors: Robert J Edwards / Katayoun Mansouri / Victoria Stalls / Kartik Manne / Brian Watts / Rob Parks / Katarzyna Janowska / Sophie M C Gobeil / Megan Kopp / Dapeng Li / Xiaozhi Lu / Zekun Mu / ...Authors: Robert J Edwards / Katayoun Mansouri / Victoria Stalls / Kartik Manne / Brian Watts / Rob Parks / Katarzyna Janowska / Sophie M C Gobeil / Megan Kopp / Dapeng Li / Xiaozhi Lu / Zekun Mu / Margaret Deyton / Thomas H Oguin / Jordan Sprenz / Wilton Williams / Kevin Saunders / David Montefiori / Gregory D Sempowski / Rory Henderson / Munir Alam / Barton F Haynes / Priyamvada Acharya
Abstract: The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its Receptor Binding Domain in two conformations: receptor-accessible "up" or receptor-inaccessible ...The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its Receptor Binding Domain in two conformations: receptor-accessible "up" or receptor-inaccessible "down" conformations. Here, we report that the commonly used stabilized S ectodomain construct "2P" is sensitive to cold temperature, and that this cold sensitivity is resolved in a "down" state stabilized spike. Our results will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.
History
DepositionNov 4, 2020-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateNov 18, 2020-
Current statusNov 18, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_22934.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map of rS2d-hexapro SARS-CoV-2 spike ectodomain
Voxel sizeX=Y=Z: 4.02 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.10771254 - 0.16261765
Average (Standard dev.)0.0005104871 (±0.012216224)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 385.91998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.024.024.02
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z385.920385.920385.920
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean-0.1080.1630.001

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Supplemental data

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Mask #1

Fileemd_22934_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Unsharpened map of rS2d-hexapro SARS-CoV-2 spike ectodomain

Fileemd_22934_additional_1.map
AnnotationUnsharpened map of rS2d-hexapro SARS-CoV-2 spike ectodomain
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half-map 2 of rS2d-hexapro SARS-CoV-2 spike ectodomain

Fileemd_22934_half_map_1.map
AnnotationHalf-map 2 of rS2d-hexapro SARS-CoV-2 spike ectodomain
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half-map 1 of rS2d-hexapro SARS-CoV-2 spike ectodomain

Fileemd_22934_half_map_2.map
AnnotationHalf-map 1 of rS2d-hexapro SARS-CoV-2 spike ectodomain
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : SARS-CoV-2 rS2d Down State Spike Protein Trimer

EntireName: SARS-CoV-2 rS2d Down State Spike Protein Trimer
Components
  • Complex: SARS-CoV-2 rS2d Down State Spike Protein Trimer
    • Protein or peptide: rS2d-hexapro SARS-CoV-2 spike ectodomain

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Supramolecule #1: SARS-CoV-2 rS2d Down State Spike Protein Trimer

SupramoleculeName: SARS-CoV-2 rS2d Down State Spike Protein Trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293F / Recombinant plasmid: p-alpha-H
Molecular weightTheoretical: 481.8 KDa

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Macromolecule #1: rS2d-hexapro SARS-CoV-2 spike ectodomain

MacromoleculeName: rS2d-hexapro SARS-CoV-2 spike ectodomain / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human)
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 GVCPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV VVLSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG VLTESNKKFL PFQQFGRDIA DTTDAVRDPQ TLEILDITPC SFGGVSVITP GTNTSNQVAV LYQDVNCTEV PVAIHADQLT PTWRVYSTGS NVFQTRAGCL IGAEHVNNSY ECDIPIGAGI CASYQTQTNS PGSASSVASQ SIIAYTMSLG AENSVAYSNN SIAIPTNFTI SVTTEILPVS MTKTSVDCTM YICGDSTECS NLLLQYGSFC TQLNRALTGI AVEQDKNTQE VFAQVKQIYK TPPIKDFGGF NFSQILPDPS KPSKRSPIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFGA GPALQIPFPM QMAYRFNGIG VTQNVLYENQ KLIANQFNSA IGKIQDSLSS TPSALGKLQD VVNQNAQALN TLVKQLSSNF GAISSVLNDI LSRLCPPEAE VQIDRLITGR LQSLQTYVTQ QLIRAAEIRA SANLAATKMS ECVLGQSKRV DFCGKGYHLM SFPQSAPHGV VFLHVTYVPA QEKNFTTAPA ICHDGKAHFP REGVFVSNGT HWFVTQRNFY EPQIITTDNT FVSGNCDVVI GIVNNTVYDP LQPELDSFKE ELDKYFKNHT SPDVDLGDIS GINASVVNIQ KEIDRLNEVA KNLNESLIDL QELGKYEQ

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
20.0 mMHEPES buffer
5.0 g/dLglycerol
StainingType: NEGATIVE / Material: Uranyl Formate
Details: Samples were diluted to 0.1 mg/mL in 20 mM HEPES buffer, pH 7.4, with 5% glycerol, 150 mM NaCl, and 7.5 mM glutaraldehyde. After 5 minute incubation at room temperature, sufficient 1 M Tris ...Details: Samples were diluted to 0.1 mg/mL in 20 mM HEPES buffer, pH 7.4, with 5% glycerol, 150 mM NaCl, and 7.5 mM glutaraldehyde. After 5 minute incubation at room temperature, sufficient 1 M Tris stock, pH 7.4, was added to a final concentration of 75 mM Tris to quench unreacted glutaraldehyde, and was then incubated 5 minutes. Sample was then applied to a carbon film over 400 mesh copper EM grids that had been glow-discharged, incubated 1 minute, and then stained with 2% uranyl formate.
GridModel: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.05 nm / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeFEI/PHILIPS EM420
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 82000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Image recordingFilm or detector model: OTHER / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Digitization - Sampling interval: 33.0 µm / Number grids imaged: 1 / Number real images: 145 / Average exposure time: 0.5 sec. / Average electron dose: 32.0 e/Å2

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Image processing

Particle selectionNumber selected: 140561
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.8)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

Details: Map of PDB 6VSB was generated within Chimera using the molmap function at 15 Angstrom resolution and 4.02 Angstrom grid spacing. This map was low-pass filtered to 60 Angstrom before initiating refinements.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 6 / Avg.num./class: 18762 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 10.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 52516
FSC plot (resolution estimation)

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