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- EMDB-3869: The structure of Ebola virus nucleoprotein (residues 1-450) in nu... -

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Basic information

Entry
Database: EMDB / ID: EMD-3869
TitleThe structure of Ebola virus nucleoprotein (residues 1-450) in nucleocapsid-like assemblies
Map dataSubtomogram averaging of recombinantly expressed Ebola Nucleoprotein (NP), residues 1-450
Sample
  • Virus: Ebola virus - Mayinga, Zaire, 1976
    • Protein or peptide: Nucleoprotein
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 (strain Mayinga-76) / Ebola virus - Mayinga, Zaire, 1976
Methodsubtomogram averaging / cryo EM / Resolution: 6.6 Å
AuthorsWan W / Kolesnikova L / Clarke M / Koehler A / Noda T / Becker S / Briggs JAG
Funding support Germany, 3 items
OrganizationGrant numberCountry
European Research CouncilERC-CoG-648432 MEMBRANEFUSION Germany
German Research FoundationSonderforschungsbereich 1021 Germany
European Molecular Biology OrganizationALTF 748-2014 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, 2017-
Header (metadata) releaseNov 8, 2017-
Map releaseNov 8, 2017-
UpdateDec 4, 2019-
Current statusDec 4, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ehl
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6ehl
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3869.png

Downloads & links

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Map

FileDownload / File: emd_3869.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram averaging of recombinantly expressed Ebola Nucleoprotein (NP), residues 1-450
Voxel sizeX=Y=Z: 1.78 Å
Density
Contour LevelBy AUTHOR: 2.5 / Movie #1: 2.5
Minimum - Maximum-3.2473512 - 8.931272
Average (Standard dev.)-0.022757614 (±1.0010257)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 341.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.781.781.78
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z341.760341.760341.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-3.2478.931-0.023

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Supplemental data

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Sample components

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Entire : Ebola virus - Mayinga, Zaire, 1976

EntireName: Ebola virus - Mayinga, Zaire, 1976
Components
  • Virus: Ebola virus - Mayinga, Zaire, 1976
    • Protein or peptide: Nucleoprotein

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Supramolecule #1: Ebola virus - Mayinga, Zaire, 1976

SupramoleculeName: Ebola virus - Mayinga, Zaire, 1976 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 128952 / Sci species name: Ebola virus - Mayinga, Zaire, 1976 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host systemOrganism: Homo sapiens (human) / Recombinant cell: HEK 293T
Virus shellShell ID: 1 / Name: Nucleocapsid / Diameter: 280.0 Å

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Macromolecule #1: Nucleoprotein

MacromoleculeName: Nucleoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Zaire ebolavirus (strain Mayinga-76) / Strain: Mayinga-76
Molecular weightTheoretical: 83.3875 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDSRPQKIWM APSLTESDMD YHKILTAGLS VQQGIVRQRV IPVYQVNNLE EICQLIIQAF EAGVDFQESA DSFLLMLCLH HAYQGDYKL FLESGAVKYL EGHGFRFEVK KRDGVKRLEE LLPAVSSGKN IKRTLAAMPE EETTEANAGQ FLSFASLFLP K LVVGEKAC ...String:
MDSRPQKIWM APSLTESDMD YHKILTAGLS VQQGIVRQRV IPVYQVNNLE EICQLIIQAF EAGVDFQESA DSFLLMLCLH HAYQGDYKL FLESGAVKYL EGHGFRFEVK KRDGVKRLEE LLPAVSSGKN IKRTLAAMPE EETTEANAGQ FLSFASLFLP K LVVGEKAC LEKVQRQIQV HAEQGLIQYP TAWQSVGHMM VIFRLMRTNF LIKFLLIHQG MHMVAGHDAN DAVISNSVAQ AR FSGLLIV KTVLDHILQK TERGVRLHPL ARTAKVKNEV NSFKAALSSL AKHGEYAPFA RLLNLSGVNN LEHGLFPQLS AIA LGVATA HGSTLAGVNV GEQYQQLREA ATEAEKQLQQ YAESRELDHL GLDDQEKKIL MNFHQKKNEI SFQQTNAMVT LRKE RLAKL TEAITAASLP KTSGHYDDDD DIPFPGPIND DDNPGHQDDD PTDSQDTTIP DVVVDPDDGS YGEYQSYSEN GMNAP DDLV LFDLDEDDED TKPVPNRSTK GGQQKNSQKG QHIEGRQTQS RPIQNVPGPH RTIHHASAPL TDNDRRNEPS GSTSPR MLT PINEEADPLD DADDETSSLP PLESDDEEQD RDGTSNRTPT VAPPAPVYRD HSEKKELPQD EQQDQDHTQE ARNQDSD NT QSEHSFEEMY RHILRSQGPF DAVLYYHMMK DEPVVFSTSD GKEYTYPDSL EEEYPPWLTE KEAMNEENRF VTLDGQQF Y WPVMNHKNKF MAILQHHQ

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statehelical array

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationFormulaName
137.0 mMNaClSodium chloridesodium chloride
2.7 mMKClpotassium chloride
10.0 mMNa2PO4disodium phosphate
1.8 mMKH2PO4monopotassium phosphate
GridModel: C-flat 2/1 3C / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: -10 eV / Energy filter - Upper energy threshold: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3708 pixel / Digitization - Dimensions - Height: 3708 pixel / Digitization - Frames/image: 1-5 / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 2.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 63 / Number images used: 488916 / Reference model: None
Software:
Namedetails
Amira (ver. 4)placing spline points
TOM Toolboxsubtomogram extraction

Details: 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.
CTF correctionSoftware:
Namedetails
CTFFIND (ver. 4)defocus determination
CTFPHASEFLIPCTF-correction

Details: CTF amplitude correction was performed during the wedge-weighted subtomogram averaging step.
Final angle assignmentType: OTHER / Software - Name: AV3
Details: Iterative angular search was performed via maximization of a modified constrained cross-correlation function.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: AV3
Details: Local resolution was estimated using moving window FSC calculations.
Number subtomograms used: 1
DetailsFrames 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.

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Atomic model buiding 1

DetailsCrystal 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.
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-6ehl:
Model of the Ebola virus nucleoprotein in recombinant nucleocapsid-like assemblies

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