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- PDB-6ehl: Model of the Ebola virus nucleoprotein in recombinant nucleocapsi... -

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Basic information

Entry
Database: PDB / ID: 6ehl
TitleModel of the Ebola virus nucleoprotein in recombinant nucleocapsid-like assemblies
ComponentsNucleoprotein
KeywordsVIRUS LIKE PARTICLE / nucleocapsid
Function / homologyEbola nucleoprotein / Ebola nucleoprotein / viral RNA genome packaging / helical viral capsid / viral nucleocapsid / host cell cytoplasm / ribonucleoprotein complex / RNA binding / Nucleoprotein
Function and homology information
Biological speciesZaire ebolavirus
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 6.6 Å
AuthorsWan, W. / Kolesnikova, L. / Clarke, M. / Koehler, A. / Noda, T. / Becker, S. / Briggs, J.A.G.
Funding support Germany, 3items
OrganizationGrant numberCountry
European Research CouncilERC-CoG-648432 MEMBRANEFUSION Germany
European Molecular Biology OrganizationALTF 748-2014 Germany
German Research FoundationSonderforschungsbereich 1021 Germany
CitationJournal: Nature / Year: 2017
Title: Structure and assembly of the Ebola virus nucleocapsid.
Authors: William Wan / Larissa Kolesnikova / Mairi Clarke / Alexander Koehler / Takeshi Noda / Stephan Becker / John A G Briggs /
Abstract: Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever. Filoviruses are within the order Mononegavirales, which also includes rabies virus, measles ...Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever. Filoviruses are within the order Mononegavirales, which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical nucleocapsid. The nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein-nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein, and cryo-electron microscopy of nucleocapsid or nucleocapsid-like structures. There have been no high-resolution reconstructions of complete mononegavirus nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus nucleocapsid within intact viruses and recombinant nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment.
History
DepositionSep 13, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 8, 2017Provider: repository / Type: Initial release
Revision 1.1Nov 22, 2017Group: Database references / Category: citation
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Dec 6, 2017Group: Database references / Category: citation
Item: _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Jan 31, 2018Group: Author supporting evidence / Data processing / Category: em_software / pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Dec 4, 2019Group: Data collection / Category: em_imaging_optics / Item: _em_imaging_optics.energyfilter_name

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Structure visualization

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Assembly

Deposited unit
A: Nucleoprotein


Theoretical massNumber of molelcules
Total (without water)83,3881
Polymers83,3881
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Nucleoprotein / / Nucleocapsid protein / Protein N / Coordinate model: Cα atoms only


Mass: 83387.500 Da / Num. of mol.: 1 / Mutation: Truncation mutant (residues 1-450)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Zaire ebolavirus (strain Mayinga-76) / Strain: Mayinga-76 / Gene: NP / Cell line (production host): HEK-293T / Production host: Homo sapiens (human) / References: UniProt: P18272

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Ebola virus - Mayinga, Zaire, 1976 / Type: VIRUS / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Ebola virus - Mayinga, Zaire, 1976
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK 293T
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Virus shellName: NucleocapsidCapsid / Diameter: 280 nm
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1137 mMsodium chlorideNaClSodium chloride1
22.7 mMpotassium chlorideKCl1
310 mMdisodium phosphateNa2PO41
41.8 mMmonopotassium phosphateKH2PO41
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat 2/1 3C
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 95 %

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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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 4500 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 2.4 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter upper: 10 eV / Energyfilter lower: -10 eV
Image scansWidth: 3708 / Height: 3708 / Movie frames/image: 5 / Used frames/image: 1-5

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Processing

EM software
IDNameVersionCategoryDetails
1Amira4volume selectionplacing spline points
2TOM Toolboxvolume selectionsubtomogram extraction
3SerialEMimage acquisition
5CTFFIND4CTF correctiondefocus determination
6CTFPHASEFLIPCTF correctionCTF-correction
9UCSF Chimeramodel fittingRigid body fit
11Cootmodel refinementBuilding missing regions
12NAMDmodel refinementFlexible fitting
14AV3final Euler assignment
16AV33D reconstruction
17MDFFmodel refinementFlexible fitting
Image processingDetails: Frames were aligned using K2Align software, based off the MotionCorr algorithm. Tomograms were reconstructed with IMOD, using stripwise CTF-correction and weighted back projection. ...Details: Frames were aligned using K2Align software, based off the MotionCorr algorithm. Tomograms were reconstructed with IMOD, using stripwise CTF-correction and weighted back projection. Subtomogram averaging was performed using scripts derived from TOM, AV3, and DYNAMO.
CTF correctionDetails: CTF amplitude correction was performed during the wedge-weighted subtomogram averaging step.
Type: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1 / Algorithm: BACK PROJECTION
Details: Local resolution was estimated using moving window FSC calculations.
Num. of class averages: 1 / Symmetry type: POINT
EM volume selectionDetails: Points along the helical axis were manually placed to define a spline. A cylindrical grid as defined at a given radius from the spline; grid spacing was chosen to provide ~4x oversampling.
Num. of tomograms: 63 / Num. of volumes extracted: 488916 / Reference model: None
Atomic model buildingProtocol: FLEXIBLE FIT
Details: Crystal structure was rigid-body fitted into density using Chimera. Missing regions of the structure was built using ideal secondary structures in coot. These were then flexibly fit using MDFF and NAMD.

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