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- PDB-6c54: Ebola nucleoprotein nucleocapsid-like assembly and the asymmetric unit -

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Basic information

Entry
Database: PDB / ID: 6c54
TitleEbola nucleoprotein nucleocapsid-like assembly and the asymmetric unit
ComponentsNucleoprotein
KeywordsRNA BINDING PROTEIN / Ebola / nucleoprotein / nucleocapsid / helical reconstruction
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
Nucleoprotein / Nucleoprotein
Similarity search - Component
Biological speciesZaire ebolavirus
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 5.8 Å
AuthorsSu, Z. / Wu, C. / Pintilie, G.D. / Chiu, W. / Amarasinghe, G.K. / Leung, D.W.
Funding support United States, 7items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103422 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM12400701 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19AI109664 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P01AI120943 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM080139 United States
Defense Threat Reduction Agency (DTRA)HDTRA1-16-1-0033 United States
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 / ...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 / ...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.
History
DepositionJan 13, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 7, 2018Provider: repository / Type: Initial release
Revision 1.1Feb 20, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Jun 9, 2021Group: Structure summary / Category: audit_author
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
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

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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
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Nucleoprotein
B: Nucleoprotein


Theoretical massNumber of molelcules
Total (without water)96,2852
Polymers96,2852
Non-polymers00
Water0
1
A: Nucleoprotein
B: Nucleoprotein
x 60


Theoretical massNumber of molelcules
Total (without water)5,777,111120
Polymers5,777,111120
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
helical symmetry operation59
2


  • Idetical with deposited unit
  • helical asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 60 / Rise per n subunits: 2.65 Å / Rotation per n subunits: -8.53 °)

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Components

#1: Protein Nucleoprotein / / eNP-2 asymmetric unit


Mass: 48142.590 Da / Num. of mol.: 2 / Fragment: UNP residues 25-457
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Zaire ebolavirus / Gene: NP / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1L7B8E4, UniProt: P18272*PLUS

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: eNP nucleocapsid-like assembly / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Zaire ebolavirus
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4 / Details: 150 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4
Buffer componentConc.: 12 mM / Name: Phosphate-buffered saline / Formula: PBS
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

MicroscopyModel: JEOL 3200FSC
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 30000 X / Cs: 4.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL 3200FSC CRYOHOLDER / Temperature (max): 86 K / Temperature (min): 86 K
Image recordingAverage exposure time: 8 sec. / Electron dose: 24 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 1266
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter upper: 30 eV / Energyfilter lower: 30 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 40 / Used frames/image: 2-32

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategoryDetails
4CTFFIND4.0.17CTF correction
7NAMD2.12model fitting
9EMAN1.9initial Euler assignmentintegrated with IHRSR
10RELION2-betafinal Euler assignment
12RELION2-beta3D reconstruction
13PHENIX1.11.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -8.53 ° / Axial rise/subunit: 2.65 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 200685
3D reconstructionResolution: 5.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 169526 / Symmetry type: HELICAL
Atomic model buildingB value: 276 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0045764
ELECTRON MICROSCOPYf_angle_d0.8597770
ELECTRON MICROSCOPYf_dihedral_angle_d5.4733491
ELECTRON MICROSCOPYf_chiral_restr0.048877
ELECTRON MICROSCOPYf_plane_restr0.0061010

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