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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 data
SampleSARS-CoV-2 rS2d Down State Spike Protein Trimer
  • 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 / Sophie M C Gobeil / Katarzyna Janowska / Dapeng Li / Xiaozhi Lu / Margaret Deyton / Jordan ...Authors: Robert J Edwards / Katayoun Mansouri / Victoria Stalls / Kartik Manne / Brian Watts / Rob Parks / Sophie M C Gobeil / Katarzyna Janowska / Dapeng Li / Xiaozhi Lu / Margaret Deyton / Jordan Sprenz / Wilton Williams / Kevin Saunders / Gregory D Sempowski / Rory Henderson / Munir Alam / Barton F Haynes / Priyamvada Acharya
Abstract: The impact of COVID-19 and the urgency to develop a vaccine against the SARS-CoV-2 virus cannot be overstated. The viral fusion spike (S) protein ectodomain is the primary target for vaccine ...The impact of COVID-19 and the urgency to develop a vaccine against the SARS-CoV-2 virus cannot be overstated. The viral fusion spike (S) protein ectodomain is the primary target for vaccine development. Here we report an unexpected cold sensitivity of a stabilized SARS-CoV-2 ectodomain construct currently being widely used for immunogen design. We found that when stored at 22 or 37 °C for 1 week, the S-protein displayed well-ordered trimeric spikes by negative stain electron microscopy. However, storage at 4 °C reduced the trimeric spikes to <10%, accompanied by decreased stability and enhanced exposure of the ACE-2 receptor binding site. Well-formed S particles could be recovered from cold-stored samples by a brief incubation at 37 °C. Our results will have broad impact on structural, functional and vaccine studies using the SARS-CoV-2 S ectodomain.
Highlights: SARS-CoV-2 S ectodomain construct, widely used for vaccine studies, exhibits cold sensitivity.Negative stain electron microscopy shows disintegration of spike structure upon storage at 4 ...Highlights: SARS-CoV-2 S ectodomain construct, widely used for vaccine studies, exhibits cold sensitivity.Negative stain electron microscopy shows disintegration of spike structure upon storage at 4 °C.Differential scanning calorimetry measurements confirm destabilization by cold.Cold storage alters antigenicity of SARS-CoV-2 spike.Brief incubation at 37 °C restored spike integrity after cold-storage.
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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.02 Å/pix.
x 96 pix.
= 385.92 Å
4.02 Å/pix.
x 96 pix.
= 385.92 Å
4.02 Å/pix.
x 96 pix.
= 385.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

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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Segmentation: #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 / Number of components: 2

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Component #1: protein, SARS-CoV-2 rS2d Down State Spike Protein Trimer

ProteinName: SARS-CoV-2 rS2d Down State Spike Protein Trimer / Recombinant expression: No
MassTheoretical: 481.8 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Homo sapiens (human) / Vector: p-alpha-H / Cell of expression system: 293F

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Component #2: protein, rS2d-hexapro SARS-CoV-2 spike ectodomain

ProteinName: rS2d-hexapro SARS-CoV-2 spike ectodomain / Recombinant expression: No
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: negative staining
Sample solutionSpecimen conc.: 0.1 mg/mL / pH: 7.4
StainingSamples 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.
VitrificationCryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: FEI/PHILIPS EM420
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 32 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 82000 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 400.0 - 1500.0 nm
Specimen HolderModel: SIDE ENTRY, EUCENTRIC
CameraDetector: OTHER

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Image acquisition

Image acquisitionNumber of digital images: 145 / Sampling size: 33 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 52516
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / Resolution: 10.7 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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