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- EMDB-11515: Molecular pore spanning the double membranes of a mutant murine c... -

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Basic information

Entry
Database: EMDB / ID: EMD-11515
TitleMolecular pore spanning the double membranes of a mutant murine coronaviral (MHV-A59 GFP-nsp3) replication organelles.
Map dataIn situ structure of the molecular pore spanning the double-membranes of coronavirus mutant (MHV-A59 delta2-GFP3) induced replication organelles.
Sample
  • Complex: Molecular pore containing hexameric nsp3 in the double membranes of the replication organelles induced by murine coronavirus mutant (MHV-A59, delta2-GFP3 mutant).
Biological speciesMurine hepatitis virus strain A59
Methodsubtomogram averaging / cryo EM / Resolution: 40.5 Å
AuthorsBarcena M / Wolff G
Funding support Netherlands, 1 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)184.034.014 Netherlands
CitationJournal: Science / Year: 2020
Title: A molecular pore spans the double membrane of the coronavirus replication organelle.
Authors: Georg Wolff / Ronald W A L Limpens / Jessika C Zevenhoven-Dobbe / Ulrike Laugks / Shawn Zheng / Anja W M de Jong / Roman I Koning / David A Agard / Kay Grünewald / Abraham J Koster / Eric J ...Authors: Georg Wolff / Ronald W A L Limpens / Jessika C Zevenhoven-Dobbe / Ulrike Laugks / Shawn Zheng / Anja W M de Jong / Roman I Koning / David A Agard / Kay Grünewald / Abraham J Koster / Eric J Snijder / Montserrat Bárcena /
Abstract: Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. ...Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo-electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.
History
DepositionJul 29, 2020-
Header (metadata) releaseAug 19, 2020-
Map releaseAug 19, 2020-
UpdateSep 23, 2020-
Current statusSep 23, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11515.png

Downloads & links

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Map

FileDownload / File: emd_11515.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ structure of the molecular pore spanning the double-membranes of coronavirus mutant (MHV-A59 delta2-GFP3) induced replication organelles.
Voxel sizeX=Y=Z: 7.02 Å
Density
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-2.5038538 - 4.2889385
Average (Standard dev.)0.10737107 (±0.9999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-48-48-48
Dimensions969696
Spacing969696
CellA=B=C: 673.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.027.027.02
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z673.920673.920673.920
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ100115122
MAP C/R/S123
start NC/NR/NS-48-48-48
NC/NR/NS969696
D min/max/mean-2.5044.2890.107

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Supplemental data

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Sample components

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Entire : Molecular pore containing hexameric nsp3 in the double membranes ...

EntireName: Molecular pore containing hexameric nsp3 in the double membranes of the replication organelles induced by murine coronavirus mutant (MHV-A59, delta2-GFP3 mutant).
Components
  • Complex: Molecular pore containing hexameric nsp3 in the double membranes of the replication organelles induced by murine coronavirus mutant (MHV-A59, delta2-GFP3 mutant).

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Supramolecule #1: Molecular pore containing hexameric nsp3 in the double membranes ...

SupramoleculeName: Molecular pore containing hexameric nsp3 in the double membranes of the replication organelles induced by murine coronavirus mutant (MHV-A59, delta2-GFP3 mutant).
type: complex / ID: 1 / Parent: 0
Details: Upon infection with murine coronavirus (MHV-A59 delta2-GFP3, doi: 10.1128/JVI.00021-14) the formation of ER derived double-membrane vesicles is induced in the host cell. The molecular pore ...Details: Upon infection with murine coronavirus (MHV-A59 delta2-GFP3, doi: 10.1128/JVI.00021-14) the formation of ER derived double-membrane vesicles is induced in the host cell. The molecular pore spans the two membranes of these viral replication organelles.
Source (natural)Organism: Murine hepatitis virus strain A59 / Organelle: virus-induced double-membrane vesicles / Location in cell: perinuclear area
Recombinant expressionOrganism: Murine hepatitis virus strain A59

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7
GridModel: Quantifoil / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 310 K / Instrument: LEICA EM GP / Details: Blotting time 15s.
Detailselectron cryo-tomography of FIB-milled lamellae

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 42000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 31 / Number images used: 154 / Method: manual picking / Software - Name: Dynamo
CTF correctionSoftware: (Name: CTFFIND, CTFPHASEFLIP)
Final 3D classificationNumber classes: 1 / Avg.num./class: 156 / Software - Name: Dynamo
Final angle assignmentType: NOT APPLICABLE / Software - Name: Dynamo
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: ALGEBRAIC (ARTS) / Resolution.type: BY AUTHOR / Resolution: 40.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Dynamo / Number subtomograms used: 156
FSC plot (resolution estimation)

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