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- EMDB-7343: Ebola nucleoprotein nucleocapsid-like assembly and the asymmetric unit -

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Entry
Database: EMDB / ID: 7343
TitleEbola nucleoprotein nucleocapsid-like assembly and the asymmetric unit
Map dataEbola nucleoprotein nucleocapsid-like assembly
SampleeNP nucleocapsid-like assembly:
Nucleoprotein
Function / homologyEbola nucleoprotein / Ebola nucleoprotein / viral RNA genome packaging / helical viral capsid / host cell cytoplasm / viral nucleocapsid / RNA binding / Nucleoprotein / Nucleoprotein
Function and homology information
SourceZaire ebolavirus
Methodhelical reconstruction / cryo EM / 5.8 Å resolution
AuthorsSu Z / Wu C
Citation
Journal: Cell / Year: 2018
Title: Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly.
Authors: Zhaoming Su / Chao Wu / Liuqing Shi / Priya Luthra / Grigore D Pintilie / Britney Johnson / Justin R Porter / Peng Ge / Muyuan Chen / Gai Liu / Thomas E Frederick / Jennifer M Binning / Gregory R Bowman / Z Hong Zhou / Christopher F Basler / Michael L Gross / Daisy W Leung / Wah Chiu / Gaya K Amarasinghe
Abstract: Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the ...Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22-α23. Biochemical, biophysical, and mutational analyses revealed that inter-eNP contacts within α22-α23 are critical for viral NC assembly and regulate viral RNA synthesis. These observations suggest that the N terminus and α22-α23 of eNP function as context-dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.
#1: Journal: Cell Rep / Year: 2015
Title: An Intrinsically Disordered Peptide from Ebola Virus VP35 Controls Viral RNA Synthesis by Modulating Nucleoprotein-RNA Interactions.
Authors: Daisy W Leung / Dominika Borek / Priya Luthra / Jennifer M Binning / Manu Anantpadma / Gai Liu / Ian B Harvey / Zhaoming Su / Ariel Endlich-Frazier / Juanli Pan / Reed S Shabman / Wah Chiu / Robert A Davey / Zbyszek Otwinowski / Christopher F Basler / Gaya K Amarasinghe
Abstract: During viral RNA synthesis, Ebola virus (EBOV) nucleoprotein (NP) alternates between an RNA-template-bound form and a template-free form to provide the viral polymerase access to the RNA template. In ...During viral RNA synthesis, Ebola virus (EBOV) nucleoprotein (NP) alternates between an RNA-template-bound form and a template-free form to provide the viral polymerase access to the RNA template. In addition, newly synthesized NP must be prevented from indiscriminately binding to noncognate RNAs. Here, we investigate the molecular bases for these critical processes. We identify an intrinsically disordered peptide derived from EBOV VP35 (NPBP, residues 20-48) that binds NP with high affinity and specificity, inhibits NP oligomerization, and releases RNA from NP-RNA complexes in vitro. The structure of the NPBP/ΔNPNTD complex, solved to 3.7 Å resolution, reveals how NPBP peptide occludes a large surface area that is important for NP-NP and NP-RNA interactions and for viral RNA synthesis. Together, our results identify a highly conserved viral interface that is important for EBOV replication and can be targeted for therapeutic development.
Validation ReportPDB-ID: 6c54

SummaryFull reportAbout validation report
DateDeposition: Jan 13, 2018 / Header (metadata) release: Feb 14, 2018 / Map release: Mar 7, 2018 / Last update: Mar 7, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6c54
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6c54
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_7343.map.gz (map file in CCP4 format, 73600 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
264 pix
2.4 Å/pix.
= 633.6 Å
264 pix
2.4 Å/pix.
= 633.6 Å
264 pix
2.4 Å/pix.
= 633.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.4 Å
Density
Contour Level:0.06 (by author), 0.06 (movie #1):
Minimum - Maximum-0.16373856 - 0.30754635
Average (Standard dev.)0.0022737961 (0.0146839395)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions264264264
Origin000
Limit263263263
Spacing264264264
CellA=B=C: 633.60004 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.42.42.4
M x/y/z264264264
origin x/y/z0.0000.0000.000
length x/y/z633.600633.600633.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS264264264
D min/max/mean-0.1640.3080.002

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Supplemental data

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Sample components

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Entire eNP nucleocapsid-like assembly

EntireName: eNP nucleocapsid-like assembly / Number of components: 2

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Component #1: protein, eNP nucleocapsid-like assembly

ProteinName: eNP nucleocapsid-like assembly / Recombinant expression: No
SourceSpecies: Zaire ebolavirus
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Nucleoprotein

ProteinName: Nucleoprotein / Recombinant expression: No
MassTheoretical: 48.14259 kDa
Source (engineered)Expression System: Zaire ebolavirus

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Experimental details

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Sample preparation

SpecimenSpecimen state: filament / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 2.65 Å / Delta phi: -8.53 deg.
Sample solutionSpecimen conc.: 5 mg/ml
Buffer solution: 150 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4
pH: 7.4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 283 K / Humidity: 100 %

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Electron microscopy imaging

ImagingMicroscope: JEOL 3200FSC
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 24 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 30000 X (nominal) / Cs: 4.7 mm / Imaging mode: BRIGHT FIELD / Energy filter: In-column Omega Filter / Energy window: 30-30 eV
Specimen HolderModel: JEOL 3200FSC CRYOHOLDER / Temperature: K ( 86 - 86 K)
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1266

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionSoftware: RELION / Resolution: 5.8 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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