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- EMDB-1421: Electron cryomicroscopy reveals different F1+F2 protein States in... -

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Basic information

Entry
Database: EMDB / ID: EMD-1421
TitleElectron cryomicroscopy reveals different F1+F2 protein States in intact parainfluenza virions.
Map dataIn-situ 3D reconstruction of the ectodomain of the PIV5 F protein determined from cryo-negative stain electron micrographs
Sample
  • Sample: F-Protein in intact Parainfluenzavirus 5
  • Protein or peptide: F-Protein
Biological speciesParainfluenza virus 5
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 15.0 Å
AuthorsLudwig K / Schade B / Boettcher C / Korte T
CitationJournal: J Virol / Year: 2008
Title: Electron cryomicroscopy reveals different F1+F2 protein States in intact parainfluenza virions.
Authors: Kai Ludwig / Boris Schade / Christoph Böttcher / Thomas Korte / Nina Ohlwein / Bolormaa Baljinnyam / Michael Veit / Andreas Herrmann /
Abstract: Electron cryomicrographs of intact parainfluenza virus 5 (PIV5) virions revealed two different surface structures, namely, a continuous layer and distinct individual spikes. The structure of these ...Electron cryomicrographs of intact parainfluenza virus 5 (PIV5) virions revealed two different surface structures, namely, a continuous layer and distinct individual spikes. The structure of these spikes reconstructed from intact virions was compared with known F ectodomain structures and was found to be different from the prefusion PIV5 F0 structure but, surprisingly, very similar to the human PIV3 F postfusion structure. Hence, we conclude that the individual F1+F2 spikes in intact PIV5 virions also correspond to the postfusion state. Since the observed fusion activity of PIV5 virions has to be associated with prefusion F1+F2 proteins, they have necessarily to be localized in the continuous surface structure. The data therefore strongly suggest that the prefusion state of the F1+F2 protein requires stabilization, most probably by the association with hemagglutinin-neuraminidase. The conversion of F1+F2 proteins from the prefusion toward the postfusion state while embedded in the virus membrane is topologically difficult to comprehend on the basis of established models and demands reconsideration of our current understanding.
History
DepositionSep 7, 2007-
Header (metadata) releaseSep 11, 2007-
Map releaseApr 7, 2008-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: -100
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: -100
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1421.gif

emd_1421.tif

Downloads & links

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Map

FileDownload / File: emd_1421.map.gz / Format: CCP4 / Size: 1.2 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationIn-situ 3D reconstruction of the ectodomain of the PIV5 F protein determined from cryo-negative stain electron micrographs
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.56 Å/pix.
x 110 pix.
= 171.6 Å		1.56 Å/pix.
x 110 pix.
= 171.6 Å		1.56 Å/pix.
x 110 pix.
= 171.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.56 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: -95.0 / Movie #1: -100
Minimum - Maximum-128.0 - 122.0
Average (Standard dev.)-124.686999999999998 (±18.075700000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-55-54-55
Dimensions110110110
Spacing110110110
CellA=B=C: 171.6 Å
α=β=γ: 90 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z1.561.561.56
M x/y/z110110110
origin x/y/z0.0000.0000.000
length x/y/z171.600171.600171.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-30-30-49
NX/NY/NZ6060100
MAP C/R/S123
start NC/NR/NS-54-55-55
NC/NR/NS110110110
D min/max/mean-128.000122.000-124.687

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Supplemental data

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Sample components

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Entire : F-Protein in intact Parainfluenzavirus 5

EntireName: F-Protein in intact Parainfluenzavirus 5
Components
  • Sample: F-Protein in intact Parainfluenzavirus 5
  • Protein or peptide: F-Protein

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Supramolecule #1000: F-Protein in intact Parainfluenzavirus 5

SupramoleculeName: F-Protein in intact Parainfluenzavirus 5 / type: sample / ID: 1000
Details: sample of fusion active virions (proven by fluorescence spectroscopy)of PIV5 (strain W3A)
Oligomeric state: F-Protein Homotrimer / Number unique components: 1
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: F-Protein

MacromoleculeName: F-Protein / type: protein_or_peptide / ID: 1 / Oligomeric state: Homotrimer / Recombinant expression: No
Source (natural)Organism: Parainfluenza virus 5 / Strain: W3A / Location in cell: viral membrane
Molecular weightExperimental: 150 KDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: PBS (150 mM NaCl, 5.8 mM NaH2PO4/Na2HPO4)
StainingType: NEGATIVE
Details: 30 seconds absorption 60 seconds staining (1% phospho-tungstic acid, pH 7.4) vitrified in liquid ethane
GridDetails: 200 mesh carbon coated collodium-supported copper grids
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: self-construction
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 ...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.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 160 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 51064 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.066 µm / Nominal defocus min: 1.501 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 92 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 100,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 4 µm / Number real images: 175 / Average electron dose: 12 e/Å2 / Bits/pixel: 8
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: MSA-based
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 3 SIGMA CUT-OFF / Software - Name: Imagic / Number images used: 5700
Detailswell resolved spike-like proteins protruding from the viral membrane suitable for single particle analysis were interactively selected

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