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- EMDB-3875: The structure of Marburg virus nucleocapsid from virions -

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Basic information

Entry
Database: EMDB / ID: EMD-3875
TitleThe structure of Marburg virus nucleocapsid from virions
Map dataSubtomogram averaging of Marburg virus nucleocapsid within intact virions
Sample
  • Virus: Lake Victoria marburgvirus - Popp
    • Protein or peptide: Marburg nucleoprotein
    • Protein or peptide: Marburg VP24
    • Protein or peptide: Marburg VP35
Biological speciesMarburg virus - Musoke, Kenya, 1980 / Marburg virus - Angola / Lake Victoria marburgvirus - Popp
Methodsubtomogram averaging / cryo EM / Resolution: 9.1 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 22, 2017-
Map releaseNov 22, 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: 1.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3875.png

Downloads & links

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Map

FileDownload / File: emd_3875.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram averaging of Marburg virus nucleocapsid within intact virions
Voxel sizeX=Y=Z: 1.78 Å
Density
Contour LevelBy AUTHOR: 1.3 / Movie #1: 1.3
Minimum - Maximum-5.4403253 - 8.994699
Average (Standard dev.)-0.024126286 (±0.9858234)
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
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-5.4408.995-0.024

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Supplemental data

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Sample components

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Entire : Lake Victoria marburgvirus - Popp

EntireName: Lake Victoria marburgvirus - Popp
Components
  • Virus: Lake Victoria marburgvirus - Popp
    • Protein or peptide: Marburg nucleoprotein
    • Protein or peptide: Marburg VP24
    • Protein or peptide: Marburg VP35

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Supramolecule #1: Lake Victoria marburgvirus - Popp

SupramoleculeName: Lake Victoria marburgvirus - Popp / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Virus was isolated from infected Huh-7 cells. Purified viruses were fixed with paraformaldehyde.
NCBI-ID: 33728 / Sci species name: Lake Victoria marburgvirus - Popp / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host systemOrganism: Homo sapiens (human) / Recombinant cell: HEK 293T
Virus shellShell ID: 1 / Name: Nucleocapsid / Diameter: 310.0 Å

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Macromolecule #1: Marburg nucleoprotein

MacromoleculeName: Marburg nucleoprotein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Marburg virus - Musoke, Kenya, 1980
SequenceString: MDLHSLLELG TKPTAPHVRN KKVILFDTNH QVSICNQIID AINSGIDLGD LLEGGLLTLC VEHYYNSDK DKFNTSPVAK YLRDAGYEFD VIKNADATRF LDVSPNEPHY SPLILALKTL E STESQRGR IGLFLSFCSL FLPKLVVGDR ASIEKALRQV TVHQEQGIVT ...String:
MDLHSLLELG TKPTAPHVRN KKVILFDTNH QVSICNQIID AINSGIDLGD LLEGGLLTLC VEHYYNSDK DKFNTSPVAK YLRDAGYEFD VIKNADATRF LDVSPNEPHY SPLILALKTL E STESQRGR IGLFLSFCSL FLPKLVVGDR ASIEKALRQV TVHQEQGIVT YPNHWLTTGH MK VIFGILR SSFILKFVLI HQGVNLVTGH DAYDSIISNS VGQTRFSGLL IVKTVLEFIL QKT DSGVTL HPLVRTSKVK NEVASFKQAL SNLARHGEYA PFARVLNLSG INNLEHGLYP QLSA IALGV ATAHGSTLAG VNVGEQYQQL REAAHDAEVK LQRRHEHQEI QAIAEDDEER KILEQ FHLQ KTEITHSQTL AVLSQKREKL ARLAAEIENN IVEDQGFKQS QNRVSQSFLN DPTPVE VTV QARPMNRPTA LPPPVDDKIE HESTEDSSSS SSFVDLNDPF ALLNEDEDTL DDSVMIP GT TSREFQGIPE PPRQSQDLNN SQGKQEDEST NRIKKQFLRY QELPPVQEDD ESEYTTDS Q ESIDQPGSDN EQGVDLPPPP LYAQEKRQDP IQHPAANPQD PFGSIGDVNG DILEPIRSP SSPSAPQEDT RMREAYELSP DFTNDEDNQQ NWPQRVVTKK GRTFLYPNDL LQTNPPESLI TALVEEYQN PVSAKELQAD WPDMSFDERR HVAMNL

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Macromolecule #2: Marburg VP24

MacromoleculeName: Marburg VP24 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Marburg virus - Musoke, Kenya, 1980
SequenceString: MAELSTRYNL PANVTENSIN LDLNSTARWI KEPSVGGWTV KWGNFVFHIP NTGMTLLHHL KSNFVVPEW QQTRNLFSHL FKNPKSTIIE PFLALRILLG VALKDQELQQ SLIPGFRSIV H MLSEWLLL EVTSAIHISP NLLGIYLTSD MFKILMAGVK NFFNKMFTLH ...String:
MAELSTRYNL PANVTENSIN LDLNSTARWI KEPSVGGWTV KWGNFVFHIP NTGMTLLHHL KSNFVVPEW QQTRNLFSHL FKNPKSTIIE PFLALRILLG VALKDQELQQ SLIPGFRSIV H MLSEWLLL EVTSAIHISP NLLGIYLTSD MFKILMAGVK NFFNKMFTLH VVNDHGKPSS IE IKLTGQQ IIITRVNMGF LVEVRRIDIE PCCGETVLSE SVVFGLVAEA VLREHSQMEK GQP LNLTQY MNSKIAI

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Macromolecule #3: Marburg VP35

MacromoleculeName: Marburg VP35 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Marburg virus - Angola
SequenceString: MWDSSYMQQV SEGLMTGKVP IDQVFGANPL EKLYKRRKPK GTVGLQCSPC LMSKATSTDD IIWDQLIVK RTLADLLIPI NRQISDIQST LSEVTTRVHE IERQLHEITP VLKMGRTLEA I SKGMSEML AKYDHLVIST GRTTAPAAAF DAYLNEHGVP PPQPAIFKDL ...String:
MWDSSYMQQV SEGLMTGKVP IDQVFGANPL EKLYKRRKPK GTVGLQCSPC LMSKATSTDD IIWDQLIVK RTLADLLIPI NRQISDIQST LSEVTTRVHE IERQLHEITP VLKMGRTLEA I SKGMSEML AKYDHLVIST GRTTAPAAAF DAYLNEHGVP PPQPAIFKDL GVAQQACSKG TM VKNATTD AADKMSKVLE LSEETFSKPN LSAKDLALLL FTHLPGNNTP FHILAQVLSK IAY KSGKSG AFLDAFHQIL SEGENAQAAL TRLSRTFDAF LGVVPPVIRV KNFQTVPRPS QKSL RAVPP NPTIDKGWVC VYSSEQGETR ALKI

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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
Details: Virus was purified into Dulbecco's modified Eagle's medium (DMEM) with 4% paraformaldehyde
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 / Average exposure time: 1.0 sec. / Average electron dose: 2.9 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 73 / Number images used: 65800 / Reference model: None
Software:
Namedetails
Amira (ver. 4)placing spline points
MATLABsubtomogram 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: TOM
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: 9.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: TOM
Details: Local resolution was estimated using moving window FSC calculations. Resolution varies from 9.1 to 13.8 Angstroms.
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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