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- EMDB-12940: SARS-CoV-2-Induced Reshaping of Subcellular Morphologies -

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Entry
Database: EMDB / ID: EMD-12940
TitleSARS-CoV-2-Induced Reshaping of Subcellular Morphologies
Map dataDual-axis tomography reconstruction of SARS-CoV-2-infected Calu-3 cells (MOI = 5) harvested 12 h after infection.
Sample
  • Tissue: Calu-3 cell line infected with SARS-CoV-2 isolate Bavpat1/2020
Biological speciesHomo sapiens (human)
Methodelectron tomography / negative staining
AuthorsCortese M / Schwab Y / Bartenschlager R / Schorb M
Funding support Germany, 3 items
OrganizationGrant numberCountry
German Research Foundation (DFG)240245660 Germany
German Research Foundation (DFG)272983813 Germany
German Research Foundation (DFG)415089553 Germany
CitationJournal: Cell Host Microbe / Year: 2020
Title: Integrative Imaging Reveals SARS-CoV-2-Induced Reshaping of Subcellular Morphologies.
Authors: Mirko Cortese / Ji-Young Lee / Berati Cerikan / Christopher J Neufeldt / Viola M J Oorschot / Sebastian Köhrer / Julian Hennies / Nicole L Schieber / Paolo Ronchi / Giulia Mizzon / Inés ...Authors: Mirko Cortese / Ji-Young Lee / Berati Cerikan / Christopher J Neufeldt / Viola M J Oorschot / Sebastian Köhrer / Julian Hennies / Nicole L Schieber / Paolo Ronchi / Giulia Mizzon / Inés Romero-Brey / Rachel Santarella-Mellwig / Martin Schorb / Mandy Boermel / Karel Mocaer / Marianne S Beckwith / Rachel M Templin / Viktoriia Gross / Constantin Pape / Christian Tischer / Jamie Frankish / Natalie K Horvat / Vibor Laketa / Megan Stanifer / Steeve Boulant / Alessia Ruggieri / Laurent Chatel-Chaix / Yannick Schwab / Ralf Bartenschlager /
Abstract: Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation-dominated cytokine response and virus-induced cell perturbation causing cell death. Here, we employ an integrative ...Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation-dominated cytokine response and virus-induced cell perturbation causing cell death. Here, we employ an integrative imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2-infected human lung epithelial cells. We report 3D electron microscopy reconstructions of whole cells and subcellular compartments, revealing extensive fragmentation of the Golgi apparatus, alteration of the mitochondrial network and recruitment of peroxisomes to viral replication organelles formed by clusters of double-membrane vesicles (DMVs). These are tethered to the endoplasmic reticulum, providing insights into DMV biogenesis and spatial coordination of SARS-CoV-2 replication. Live cell imaging combined with an infection sensor reveals profound remodeling of cytoskeleton elements. Pharmacological inhibition of their dynamics suppresses SARS-CoV-2 replication. We thus report insights into virus-induced cytopathic effects and provide alongside a comprehensive publicly available repository of 3D datasets of SARS-CoV-2-infected cells for download and smooth online visualization.
History
DepositionMay 14, 2021-
Header (metadata) releaseJun 2, 2021-
Map releaseJun 2, 2021-
UpdateJun 2, 2021-
Current statusJun 2, 2021Processing site: PDBe / Status: Released

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Structure visualization

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  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_12940.png

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Map

FileDownload / File: emd_12940.map.gz / Format: CCP4 / Size: 547 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationDual-axis tomography reconstruction of SARS-CoV-2-infected Calu-3 cells (MOI = 5) harvested 12 h after infection.
Voxel sizeX=Y=Z: 15.578 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)43.250843 (±19.93346)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin4470
Dimensions20242024140
Spacing20242024140
CellA: 31529.873 Å / B: 31529.873 Å / C: 2180.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z15.57800049407115.57800049407115.578
M x/y/z20242024140
origin x/y/z0.0000.0000.000
length x/y/z31529.87331529.8732180.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS4470
NC/NR/NS20242024140
D min/max/mean-128.000127.00043.251

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Supplemental data

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Sample components

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Entire : Calu-3 cell line infected with SARS-CoV-2 isolate Bavpat1/2020

EntireName: Calu-3 cell line infected with SARS-CoV-2 isolate Bavpat1/2020
Components
  • Tissue: Calu-3 cell line infected with SARS-CoV-2 isolate Bavpat1/2020

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Supramolecule #1: Calu-3 cell line infected with SARS-CoV-2 isolate Bavpat1/2020

SupramoleculeName: Calu-3 cell line infected with SARS-CoV-2 isolate Bavpat1/2020
type: tissue / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Organ: Lung / Tissue: Epithelial adenocarcinoma cell

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography
Aggregation statetissue

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Sample preparation

BufferpH: 7.4
StainingType: POSITIVE / Material: osmium-ferricyanide, Uranyl acetate, Lead citrate
Sugar embeddingMaterial: Epon 812
GridModel: Homemade / Material: COPPER / Support film - Material: FORMVAR
DetailsCalu-3 cells infected with SARS-CoV-2 at MOI = 5, fixed by adding 2x concentrated EM fixative (composition of the 1x fixative: 2.5% glutaraldehyde in 50 mM Na-cacodylate buffer (pH 7.4) containing 50 mM KCl, 2.6 mM MgCl2, 2.6 mM CaCl2 and 2% sucrose) to the cell culture medium (1:1) for 5 min at RT in 12 wells plates. Fixative was removed and replaced by 1x fixative for 2 h at RT. Plates were plunged in 6% formaldehyde for inactivation for 30 min at RT before being transported outside the BSL3 area. Fixative was exchanged again with 1x EM fixative and the samples were stored at 4C until further processing. Prior to embedding cells were rinsed 6 times with 100 mM Na-cacodylate for 10 min each. Subsequently, cells were post-fixed with osmium-ferricyanide (1% OsO4, 1.5% K3Fe(III)(CN)6, 0.065 M Na-cacodylate buffer) for 2 h at 4C in the dark. Further processing was done in the microwave. Cells were washed five times with dH2O for 1 min each, stained four times with 1% uranyl acetate in dH2O for 2 min each, rinsed three times with dH2O for 1 min. Dehydration with an ethanol series (50%, 70%, 90% and 4x 100%) was then performed for 40 s each on ice in the microwave. Cells were infiltrated in Epon 812 resin with increasing percentages of this resin in ethanol (10%, 30%, 50%, 70%, 90% and 2x 100%) for 3 min each in the microwave. The coverslips with the cells facing down were placed on a BEEM capsule filled with Epon 812 resin. Beem capsule and coverslip were turned upside down and polymerized at 60C. After one day the glass coverslips were removed from the blocks that were incubated for 2 more days at 60C. Grids were post-stained with Uranyl acetate and lead citrate.
SectioningUltramicrotomy - Instrument: Leica EM UC7 / Ultramicrotomy - Temperature: 293 K / Ultramicrotomy - Final thickness: 200 nm

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus min: 0.5 µm / Nominal magnification: 15500
Sample stageSpecimen holder model: FISCHIONE INSTRUMENTS DUAL AXIS TOMOGRAPHY HOLDER
Image recordingFilm or detector model: OTHER / Digitization - Dimensions - Width: 2024 pixel / Digitization - Dimensions - Height: 2024 pixel / Number grids imaged: 1 / Number real images: 242 / Average exposure time: 0.2 sec. / Average electron dose: 3.6 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.10) / Number images used: 242
DetailsGatan OneView

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