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- EMDB-24600: Tomogram of SARS-CoV-2 spike-bearing virus-like particles (VLPs) ... -

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Basic information

Entry
Database: EMDB / ID: EMD-24600
TitleTomogram of SARS-CoV-2 spike-bearing virus-like particles (VLPs) interacting with hACE2-bearing extracellular vesicles (tEVs), showing various intermediate states of the SARS-CoV-2 spike protein (Fig. 2K,L, and Supp. Movie 2 of the manuscript Marcink et. al 2021,).
Map dataTomogram of SARS-CoV-2 spike-bearing virus-like particles (VLPs) interacting with hACE2-bearing extracellular vesicles (tEVs), showing various intermediate states of the SARS-CoV-2 spike protein (Fig. 2K,L, and Supp. Movie 2 of the manuscript Marcink et. al 2021,).
Sample
  • Complex: Virus-like particles and extracellular vesicles
    • Complex: hACE2-bearing target extracellular vesicles(tEVs)
    • Complex: SARS-CoV-2 spike-bearing virus-like particles (VLPs)
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Homo sapiens HEK293-T (human)
Methodelectron tomography / cryo EM
AuthorsMarcink TC / Porotto M / des Georges A / Moscona A
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)F32AI152275 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI114736 United States
CitationJournal: Sci Adv / Year: 2022
Title: Intermediates in SARS-CoV-2 spike-mediated cell entry.
Authors: Tara C Marcink / Thomas Kicmal / Emily Armbruster / Zhening Zhang / Gillian Zipursky / Kate L Golub / Mohab Idris / Jonathan Khao / Jennifer Drew-Bear / Gael McGill / Tom Gallagher / Matteo ...Authors: Tara C Marcink / Thomas Kicmal / Emily Armbruster / Zhening Zhang / Gillian Zipursky / Kate L Golub / Mohab Idris / Jonathan Khao / Jennifer Drew-Bear / Gael McGill / Tom Gallagher / Matteo Porotto / Amédée des Georges / Anne Moscona /
Abstract: SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by ...SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo-electron microscopy and cryo-electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo-electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein-directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors.
History
DepositionJul 31, 2021-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateAug 31, 2022-
Current statusAug 31, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24600.png

Downloads & links

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Map

FileDownload / File: emd_24600.map.gz / Format: CCP4 / Size: 702.4 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationTomogram of SARS-CoV-2 spike-bearing virus-like particles (VLPs) interacting with hACE2-bearing extracellular vesicles (tEVs), showing various intermediate states of the SARS-CoV-2 spike protein (Fig. 2K,L, and Supp. Movie 2 of the manuscript Marcink et. al 2021,).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.48 Å/pix.
x 250 pix.
= 1621. Å		6.48 Å/pix.
x 1440 pix.
= 9336.96 Å		6.48 Å/pix.
x 1023 pix.
= 6633.132 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 6.484 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-1818.0 - 767.0
Average (Standard dev.)87.285034 (±35.790585)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-208209-75
Dimensions14401023250
Spacing10231440250
CellA: 6633.1323 Å / B: 9336.96 Å / C: 1621.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Virus-like particles and extracellular vesicles

EntireName: Virus-like particles and extracellular vesicles
Components
  • Complex: Virus-like particles and extracellular vesicles
    • Complex: hACE2-bearing target extracellular vesicles(tEVs)
    • Complex: SARS-CoV-2 spike-bearing virus-like particles (VLPs)

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Supramolecule #1: Virus-like particles and extracellular vesicles

SupramoleculeName: Virus-like particles and extracellular vesicles / type: complex / ID: 1 / Parent: 0

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Supramolecule #2: hACE2-bearing target extracellular vesicles(tEVs)

SupramoleculeName: hACE2-bearing target extracellular vesicles(tEVs) / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2

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Supramolecule #3: SARS-CoV-2 spike-bearing virus-like particles (VLPs)

SupramoleculeName: SARS-CoV-2 spike-bearing virus-like particles (VLPs) / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Homo sapiens HEK293-T (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Support film - Film thickness: 300.0 nm
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Sigma Aldrich / Diameter: 5 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 2.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 8.0 µm / Calibrated defocus min: 5.0 µm / Calibrated magnification: 53000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: eTomo (ver. 4.10.52) / Number images used: 37
CTF correctionSoftware: (Name: Warp (ver. 1.0.9), eTomo (ver. 4.10.52))

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