|Entry||Database: PDB / ID: 6ehm|
|Title||Model of the Ebola virus nucleocapsid subunit from recombinant virus-like particles|
|Descriptor||Nucleoprotein, Membrane-associated protein VP24|
|Keywords||VIRUS LIKE PARTICLE / nucleocapsid / virus-like particle|
|Specimen source||Zaire ebolavirus (strain Mayinga-76) / virus / ZEBOV|
|Method||Electron microscopy (7.3 Å resolution / Helical array / Subtomogram averaging)|
|Authors||Wan, W. / Kolesnikova, L. / Clarke, M. / Koehler, A. / Noda, T. / Becker, S. / Briggs, J.A.G.|
|Citation||Nature, 2017, 551, 394-397|
SummaryFull reportAbout validation report
|Date||Deposition: Sep 13, 2017 / Release: Nov 8, 2017|
Downloads & links
C: Membrane-associated protein VP24
D: Membrane-associated protein VP24
Mass: 83387.500 Da / Num. of mol.: 2
Source: (gene. exp.) Zaire ebolavirus (strain Mayinga-76) / virus
References: UniProt: P18272
Mass: 28250.811 Da / Num. of mol.: 2
Source: (gene. exp.) Zaire ebolavirus (strain Mayinga-76) / virus
References: UniProt: Q05322
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: HELICAL ARRAY / Reconstruction method: SUBTOMOGRAM AVERAGING|
|Component||Name: Ebola virus - Mayinga, Zaire, 1976 / Type: VIRUS|
Details: Recombinantly expressed virus-like particles produced by expression of nucleoprotein, VP24, VP35, VP40.
Entity ID: 1, 2 / Source: RECOMBINANT
|Source (natural)||Organism: Ebola virus - Mayinga, Zaire, 1976|
|Source (recombinant)||Cell: HEK 293T / Organism: Homo sapiens|
|Details of virus||Empty: NO / Enveloped: YES / Virus isolate: STRAIN / Virus type: VIRUS-LIKE PARTICLE|
|Virus shell||Name: Nucleocapsid / Diameter: 280 Å|
|Buffer solution||pH: 7.4|
|Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid material: COPPER / Grid mesh size: 300 / Grid type: C-flat 2/1 3C|
|Vitrification||Instrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 95 %|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELD / Nominal magnification: 81000 / Nominal defocus max: 4500 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 5 mm / Alignment procedure: ZEMLIN TABLEAU|
|Specimen holder||Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Average exposure time: 2.3 sec. / Electron dose: 3.4 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k)|
|EM imaging optics||Energyfilter name: Gatan Quantum 967 LS / Energyfilter upper: 10 eV / Energyfilter lower: -10 eV|
|Image scans||Dimension width: 3708 / Dimension height: 3708 / Movie frames/image: 5 / Used frames/image: 1-5|
|Image processing||Details: Frames 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.|
|CTF correction||Details: CTF amplitude correction was performed during the wedge-weighted subtomogram averaging step.|
Type: PHASE FLIPPING ONLY
|Symmetry||Point symmetry: C1|
|3D reconstruction||Resolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 1 / Algorithm: BACK PROJECTION|
Details: Local resolution was estimated using moving window FSC calculations. Resolution varies from 7.3 to 15.2 Angstroms.
Number of class averages: 1 / Symmetry type: POINT
|EM volume selection||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.|
Number of tomograms: 63 / Number of volumes extracted: 379428 / Reference model: None
|Atomic model building||Details: Nucleoprotein model from Ebola virus nucleoprotein 1-450 was first rigid body fitted into the inner nucleoprotein densities. Densities were then subtracted, and VP24 pdb was then fit into remaining densities. All models were rigid-body fitted using UCSF Chimera.|
Ref protocol: RIGID BODY FIT
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