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- PDB-8ust: In-virion structure of Ebola virus nucleocapsid-like assemblies f... -

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Basic information

Entry
Database: PDB / ID: 8ust
TitleIn-virion structure of Ebola virus nucleocapsid-like assemblies from recombinant virus-like particles (nucleoprotein, VP24,VP35,VP40)
Components
  • Membrane-associated protein VP24
  • Nucleoprotein
  • Polymerase cofactor VP35
  • RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')
KeywordsVIRAL PROTEIN/RNA / VIRAL PROTEIN / nucleoprotein / nucleocapsid / Ebola virus / EBOV / filovirus / subtomogram averaging / in situ / VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


host cell endomembrane system / symbiont-mediated suppression of host defenses / symbiont-mediated suppression of host RNAi-mediated antiviral immune response / negative regulation of miRNA-mediated gene silencing / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IKBKE activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF7 activity / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class II / positive regulation of protein sumoylation / viral RNA genome packaging / helical viral capsid ...host cell endomembrane system / symbiont-mediated suppression of host defenses / symbiont-mediated suppression of host RNAi-mediated antiviral immune response / negative regulation of miRNA-mediated gene silencing / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IKBKE activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF7 activity / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class II / positive regulation of protein sumoylation / viral RNA genome packaging / helical viral capsid / viral transcription / molecular sequestering activity / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity / viral genome replication / viral budding from plasma membrane / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / viral nucleocapsid / symbiont-mediated suppression of host toll-like receptor signaling pathway / host cell cytoplasm / symbiont-mediated suppression of host innate immune response / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / ribonucleoprotein complex / negative regulation of gene expression / host cell plasma membrane / virion membrane / structural molecule activity / RNA binding / membrane
Similarity search - Function
Filovirus membrane-associated VP24 / Filovirus membrane-associated protein VP24 / Ebola nucleoprotein / Ebola nucleoprotein / Filoviruses VP35 interferon inhibitory domain, beta-sheet subdomain / Filoviridae VP35 protein / Filoviruses VP35 interferon inhibitory domain / Filoviruses VP35 interferon inhibitory domain, helical subdomain / Filoviridae VP35 / Filoviruses VP35 interferon inhibitory domain profile.
Similarity search - Domain/homology
RNA / Nucleoprotein / Polymerase cofactor VP35 / Membrane-associated protein VP24
Similarity search - Component
Biological speciesEbola virus - Mayinga
Zaire (virus)
1976
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 7.3 Å
AuthorsWatanabe, R. / Zyla, D. / Saphire, E.O.
Funding support1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Database references / Structure summary / Category: citation / pdbx_entry_details
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nucleoprotein
B: Nucleoprotein
C: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')
D: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')
E: Nucleoprotein
I: Membrane-associated protein VP24
J: Membrane-associated protein VP24
K: Polymerase cofactor VP35
F: Polymerase cofactor VP35


Theoretical massNumber of molelcules
Total (without water)385,3319
Polymers385,3319
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Nucleoprotein


Mass: 83387.500 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: The N-terminal part of nucleoprotein / Source: (gene. exp.) Ebola virus - Mayinga, Zaire, 1976 / Gene: NP / Production host: Homo sapiens (human) / References: UniProt: P18272
#2: RNA chain RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')


Mass: 1930.277 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Sample RNA sequence / Source: (gene. exp.) Zaire (virus) / Production host: Homo sapiens (human)
#3: Protein Membrane-associated protein VP24 / Ebola VP24 / eVP24


Mass: 28250.811 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: VP24 / Source: (gene. exp.) Ebola virus - Mayinga, Zaire, 1976 / Gene: VP24 / Production host: Homo sapiens (human) / References: UniProt: Q05322
#4: Protein Polymerase cofactor VP35


Mass: 37403.277 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: C-terminal domain of VP35 / Source: (gene. exp.) Ebola virus - Mayinga, Zaire, 1976 / Gene: VP35 / Production host: Homo sapiens (human) / References: UniProt: Q05127
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: subtomogram averaging

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Ebola virus - Mayinga, Zaire, 1976VIRUS4 virus proteins, NP, VP24, VP35, VP40 were expressed from a plasmid in HEK 293T cells Virus-like-particles were isolated from the supernatant.all0RECOMBINANT
2NP core with RNACOMPLEX#1-#21RECOMBINANT
3Outer NP with VP35 NP-binding peptideCOMPLEX#1, #41RECOMBINANT
4VP24 second layer of nucleocapsidCOMPLEX#31RECOMBINANT
5VP35 CTDCOMPLEX#41RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
22
31NO
51NO
61NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Ebola virus - Mayinga, Zaire, 1976128952
32Ebola virus - Mayinga, Zaire, 1976128952
43Ebola virus - Mayinga, Zaire, 1976128952
65Ebola virus - Mayinga, Zaire, 1976128952
75Ebola virus - Mayinga, Zaire, 1976128952
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
65Homo sapiens (human)9606
75Homo sapiens (human)9606
Details of virus
IDEntity assembly-IDEmptyEnvelopedIsolateType
11NOYESOTHERVIRUS-LIKE PARTICLE
22
33
55
65
Natural host
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens9606
22
33
55
65
Buffer solutionpH: 7.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Viral-like-particles isolated from cells expressing NP,VP24,VP35 and VP40
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 4500 nm / Nominal defocus min: 2000 nm
Image recordingElectron dose: 1.6 e/Å2 / Avg electron dose per subtomogram: 100 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35366 / Symmetry type: POINT
EM volume selectionNum. of tomograms: 56 / Num. of volumes extracted: 35366

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