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- PDB-5z9w: Ebola virus nucleoprotein-RNA complex -

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Basic information

Entry
Database: PDB / ID: 5z9w
TitleEbola virus nucleoprotein-RNA complex
Components
  • Ebolavirus nucleoprotein (residues 19-406)
  • RNA (6-MER)
KeywordsVIRAL PROTEIN / ebolavirus / RNA / nucleoprotein / nucleocapsid / helical
Function / homology
Function and homology information


viral RNA genome packaging / helical viral capsid / viral nucleocapsid / host cell cytoplasm / ribonucleoprotein complex / RNA binding
Similarity search - Function
Ebola nucleoprotein / Ebola nucleoprotein
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsSugita, Y. / Matsunami, H. / Kawaoka, Y. / Noda, T. / Wolf, M.
Funding support Japan, 9items
OrganizationGrant numberCountry
the Ministry of Education, Culture, Science, Sports, and Technology26892028 Japan
Japan Science and Technology AgencyJPMJPR13L9 Japan
Japan Agency for Medical Research and Development17fk0108128h0001 Japan
the Ministry of Education, Culture, Science, Sports, and Technology16H06429 Japan
the Ministry of Education, Culture, Science, Sports, and Technology16K21723 Japan
the Ministry of Education, Culture, Science, Sports, and Technology16H06434 Japan
Japan Society for the Promotion of Science Japan
Daiichi Sankyo Foundation of Life Science Japan
Japan Agency for Medical Research and Development Japan
CitationJournal: Nature / Year: 2018
Title: Cryo-EM structure of the Ebola virus nucleoprotein-RNA complex at 3.6 Å resolution.
Authors: Yukihiko Sugita / Hideyuki Matsunami / Yoshihiro Kawaoka / Takeshi Noda / Matthias Wolf /
Abstract: Ebola virus causes haemorrhagic fever with a high fatality rate in humans and non-human primates. It belongs to the family Filoviridae in the order Mononegavirales, which are viruses that contain ...Ebola virus causes haemorrhagic fever with a high fatality rate in humans and non-human primates. It belongs to the family Filoviridae in the order Mononegavirales, which are viruses that contain linear, non-segmented, negative-sense, single-stranded genomic RNA. The enveloped, filamentous virion contains the nucleocapsid, consisting of the helical nucleoprotein-RNA complex, VP24, VP30, VP35 and viral polymerase. The nucleoprotein-RNA complex acts as a scaffold for nucleocapsid formation and as a template for RNA replication and transcription by condensing RNA into the virion. RNA binding and nucleoprotein oligomerization are synergistic and do not readily occur independently. Although recent cryo-electron tomography studies have revealed the overall architecture of the nucleocapsid core, there has been no high-resolution reconstruction of the nucleocapsid. Here we report the structure of a recombinant Ebola virus nucleoprotein-RNA complex expressed in mammalian cells without chemical fixation, at near-atomic resolution using single-particle cryo-electron microscopy. Our structure reveals how the Ebola virus nucleocapsid core encapsidates its viral genome, its sequence-independent coordination with RNA by nucleoprotein, and the dynamic transition between the RNA-free and RNA-bound states. It provides direct structural evidence for the role of the N terminus of nucleoprotein in subunit oligomerization, and for the hydrophobic and electrostatic interactions that lead to the formation of the helical assembly. The structure is validated as representative of the native biological assembly of the nucleocapsid core by consistent dimensions and symmetry with the full virion. The atomic model provides a detailed mechanistic basis for understanding nucleocapsid assembly and highlights key structural features that may serve as targets for anti-viral drug development.
History
DepositionFeb 5, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 24, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 7, 2018Group: Data collection / Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed
Revision 1.2Nov 14, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title
Revision 1.3Nov 6, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.4Dec 16, 2020Group: Author supporting evidence / Category: em_helical_entity / Item: _em_helical_entity.axial_symmetry
Revision 1.5Mar 27, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list / refine
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type / _refine.ls_d_res_high

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Structure visualization

Movie
  • Biological unit as representative helical assembly
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-6903
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Ebolavirus nucleoprotein (residues 19-406)
R: RNA (6-MER)


Theoretical massNumber of molelcules
Total (without water)45,2882
Polymers45,2882
Non-polymers00
Water0
1
A: Ebolavirus nucleoprotein (residues 19-406)
R: RNA (6-MER)
x 51


Theoretical massNumber of molelcules
Total (without water)2,309,694102
Polymers2,309,694102
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
helical symmetry operation50
SymmetryHelical symmetry: (Num. of operations: 51 )

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Components

#1: Protein Ebolavirus nucleoprotein (residues 19-406)


Mass: 43496.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell (production host): epithelial / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / Tissue (production host): embryonic kidney / References: UniProt: P18272*PLUS
#2: RNA chain RNA (6-MER)


Mass: 1792.037 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Ebolavirus nucleoprotein RNA complex (biological assembly)
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.166 MDa / Experimental value: NO
Source (natural)Organism: Ebola virus - Mayinga, Zaire, 1976
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293T
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris-HClTrisC4H12ClNO31
2150 mMsodium chlorideNaClSodium chloride1
31 mMEDTAEthylenediaminetetraacetic acidC10H12O8CaN2Na2x2H2O1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Gatan Solarus / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 K / Details: 3 second blot, 2.5uL

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: nanoprobe, parallel beam illumination
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Calibrated magnification: 47619 X / Nominal defocus max: 4000 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 4000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 77 K / Residual tilt: 0.1 mradians
Image recordingAverage exposure time: 15 sec. / Electron dose: 105 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2467
Details: frame alignment and dose weighting using motioncor2
EM imaging opticsEnergyfilter name: GIF Quantum LS
Image scansSampling size: 5 µm / Width: 7676 / Height: 7420 / Movie frames/image: 75 / Used frames/image: 1-75

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1EMAN22.1particle selectione2helixboxer.py
2Leginon3.2image acquisition
4CTFFIND4.1CTF correction
7Coot0.8.7model fitting
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignment
11RELION2.1classification
12RELION2.13D reconstruction
13PHENIX1.13rc2-2981model refinementphenix.real_space_refine
Image processingDetails: frame alignment and integration with motioncor2 incl. dose weighting and 2x Fourier cropping
CTF correctionDetails: deconvolution in RELION / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -14.73 ° / Axial rise/subunit: 3.01 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 232490
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 117552 / Algorithm: FOURIER SPACE / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementHighest resolution: 3.6 Å

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