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- EMDB-42515: Intracellular Ebola nucleocapsid-like structure obtained from cel... -

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Entry
Database: EMDB / ID: EMD-42515
TitleIntracellular Ebola nucleocapsid-like structure obtained from cells expressing NP, VP24 and VP35 at 17.6 angstrom
Map dataintracellular Ebola virus nucelocapsid from cells expressing NP, VP24 and VP35
Sample
  • Virus: Ebola virus - Mayinga, Zaire, 1976
KeywordsVIRAL PROTEIN / nucleoprotein / nucleocapsid / Ebola virus / EBOV / filovirus / subtomogram averaging / cryo-ET / FIB / intracellular / in situ
Biological speciesEbola virus - Mayinga, Zaire, 1976
Methodsubtomogram averaging / cryo EM / Resolution: 17.57 Å
AuthorsWatanabe R / Zyla D / Saphire EO
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell / Year: 2024
Title: Intracellular Ebola virus nucleocapsid assembly revealed by in situ cryo-electron tomography.
Authors: Reika Watanabe / Dawid Zyla / Diptiben Parekh / Connor Hong / Ying Jones / Sharon L Schendel / William Wan / Guillaume Castillon / Erica Ollmann Saphire /
Abstract: Filoviruses, including the Ebola and Marburg viruses, cause hemorrhagic fevers with up to 90% lethality. The viral nucleocapsid is assembled by polymerization of the nucleoprotein (NP) along the ...Filoviruses, including the Ebola and Marburg viruses, cause hemorrhagic fevers with up to 90% lethality. The viral nucleocapsid is assembled by polymerization of the nucleoprotein (NP) along the viral genome, together with the viral proteins VP24 and VP35. We employed cryo-electron tomography of cells transfected with viral proteins and infected with model Ebola virus to illuminate assembly intermediates, as well as a 9 Å map of the complete intracellular assembly. This structure reveals a previously unresolved third and outer layer of NP complexed with VP35. The intrinsically disordered region, together with the C-terminal domain of this outer layer of NP, provides the constant width between intracellular nucleocapsid bundles and likely functions as a flexible tether to the viral matrix protein in the virion. A comparison of intracellular nucleocapsids with prior in-virion nucleocapsid structures reveals that the nucleocapsid further condenses vertically in the virion. The interfaces responsible for nucleocapsid assembly are highly conserved and offer targets for broadly effective antivirals.
History
DepositionOct 29, 2023-
Header (metadata) releaseOct 2, 2024-
Map releaseOct 2, 2024-
UpdateOct 2, 2024-
Current statusOct 2, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42515.png

Downloads & links

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Map

FileDownload / File: emd_42515.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationintracellular Ebola virus nucelocapsid from cells expressing NP, VP24 and VP35
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.37 Å/pix.
x 72 pix.
= 386.64 Å		5.37 Å/pix.
x 72 pix.
= 386.64 Å		5.37 Å/pix.
x 72 pix.
= 386.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 5.37 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.288
Minimum - Maximum-0.57818794 - 0.8626646
Average (Standard dev.)0.02965617 (±0.14129469)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions727272
Spacing727272
CellA=B=C: 386.63998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42515_msk_1.map
Projections & Slices
AxesZYX

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Histogram

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Half map: half map1

Fileemd_42515_half_map_1.map
Annotationhalf map1
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: half map2

Fileemd_42515_half_map_2.map
Annotationhalf map2
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ebola virus - Mayinga, Zaire, 1976

EntireName: Ebola virus - Mayinga, Zaire, 1976
Components
  • Virus: Ebola virus - Mayinga, Zaire, 1976

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Supramolecule #1: Ebola virus - Mayinga, Zaire, 1976

SupramoleculeName: Ebola virus - Mayinga, Zaire, 1976 / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 128952 / Sci species name: Ebola virus - Mayinga, Zaire, 1976 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Homo sapiens (human)

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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: 7.2
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE
DetailsFIB-milled-plunge-frozen cell expressing EBOV NP, VP24 and VP35

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 3.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.5 µm / Nominal defocus min: 4.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 17.57 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number subtomograms used: 8371
ExtractionNumber tomograms: 10 / Number images used: 39000
Final 3D classificationNumber classes: 3 / Avg.num./class: 13000 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)

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