|Entry||Database: EMDB / ID: 1421|
|Title||Electron cryomicroscopy reveals different F1+F2 protein States in intact parainfluenza virions.|
|Sample||F-Protein in intact Parainfluenzavirus 5|
|Source||Parainfluenza virus 5 / virus|
|Map data||In-situ 3D reconstruction of the ectodomain of the PIV5 F protein determined from cryo-negative stain electron micrographs|
|Method||single particle reconstruction, at 15 A resolution|
|Authors||Ludwig K / Schade B / Boettcher C / Korte T|
|Citation||J. Virol., 2008, 82, 3775-3781|
|Date||Deposition: Sep 7, 2007 / Header (metadata) release: Sep 11, 2007 / Map release: Apr 7, 2008 / Last update: Sep 7, 2007|
Downloads & links
|File||emd_1421.map.gz (map file in CCP4 format, 1301 KB)|
|Projections & slices||Size of images: |
Images are generated by Spider package.
|Voxel size||X=Y=Z: 1.56 A|
CCP4 map header:
-Entire F-Protein in intact Parainfluenzavirus 5
|Entire||Name: F-Protein in intact Parainfluenzavirus 5|
Details: sample of fusion active virions (proven by fluorescence spectroscopy)of PIV5 (strain W3A)
Number of components: 1 / Oligomeric State: F-Protein Homotrimer
|Mass||Theoretical: 150 kDa|
-Component #1: protein, F-Protein
|Protein||Name: F-Protein / Oligomeric Details: Homotrimer / Recombinant expression: No|
|Mass||Experimental: 150 kDa|
|Source||Species: Parainfluenza virus 5 / virus / Strain: W3A|
|Source (natural)||Location in cell: viral membrane|
|Sample solution||Buffer solution: PBS (150 mM NaCl, 5.8 mM NaH2PO4/Na2HPO4) / pH: 7.4|
|Support film||200 mesh carbon coated collodium-supported copper grids|
|Staining||30 seconds absorption 60 seconds staining (1% phospho-tungstic acid, pH 7.4) vitrified in liquid ethane|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE|
Method: A small vial of ethane is placed inside a larger liquid nitrogen reservoir. The grid holding a few microliters of the sample is held in place at the bottom of a plunger by the means of fine tweezers. Once the ethane in the vial is completely frozen, it needs to be slightly melted. When the liquid ethane is ready, a piece of filter paper is then pressed against the sample to blot of excess buffer, sufficient to leave a thin layer on the grid. After a redetermined time, the filter paper is removed, and the plunger is allowed to drop into the liquid ethane. Once the grid enters the liquid ethane, the sample is rapidly frozen,and the grid is transferred under liquid nitrogen to a storage box immersed liquid nitrogen for later use in the microscope.
Details: Vitrification instrument: self-construction
-Electron microscopy imaging
|Imaging||Microscope: FEI TECNAI F20|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 160 kV / Electron dose: 12 e/A2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 50000 X (nominal), 51064 X (calibrated)|
Astigmatism: objective lens astigmatism was corrected at 100,000 times magnification
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1501 - 2066 nm
|Specimen Holder||Holder: Side entry liquid nitrogen-cooled cryo specimen holder|
Model: GATAN LIQUID NITROGEN / Temperature: 92 K
|Camera||Detector: KODAK SO-163 FILM|
|Image acquisition||Number of digital images: 175 / Scanner: PRIMESCAN / Sampling size: 4 microns / Bit depth: 8|
|Processing||Method: single particle reconstruction / Number of projections: 5700|
Details: well resolved spike-like proteins protruding from the viral membrane suitable for single particle analysis were interactively selected
Applied symmetry: C3 (3 fold cyclic)
|3D reconstruction||Algorithm: Common lines / Software: Imagic / CTF correction: MSA-based / Resolution: 15 A / Resolution method: FSC 3 SIGMA|
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