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- EMDB-2374: cryo-electron microscopy of Sindbis-liposome complex at pH 6.4 (t... -

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Basic information

Entry
Database: EMDB / ID: EMD-2374
Titlecryo-electron microscopy of Sindbis-liposome complex at pH 6.4 (the group of 5-fold reconstruction)
Map datareconstruction of Sindbis virus when a liposome associates to its 50-fold axis. 5-fold symmetry was applied to the reconstruction map.
Sample
  • Sample: Sindbis virus - liposome complex at pH 6.4
  • Virus: Sindbis virus
Keywordscryo-electron microscopy / 3D reconstruction / Sindbis virus / membrane fusion / low pH
Biological speciesSindbis virus
Methodsingle particle reconstruction / cryo EM / Resolution: 14.5 Å
AuthorsCao S / Zhang W
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: Characterization of an early-stage fusion intermediate of Sindbis virus using cryoelectron microscopy.
Authors: Sheng Cao / Wei Zhang /
Abstract: The sequential steps in the alphavirus membrane fusion pathway have been postulated based on the prefusion and postfusion crystal structures of the viral fusion protein E1 in conjunction with ...The sequential steps in the alphavirus membrane fusion pathway have been postulated based on the prefusion and postfusion crystal structures of the viral fusion protein E1 in conjunction with biochemical studies. However, the molecular structures of the hypothesized fusion intermediates have remained obscure due to difficulties inherent in the dynamic nature of the process. We developed an experimental system that uses liposomes as the target membrane to capture Sindbis virus, a prototypical alphavirus, in its membrane-binding form at pH 6.4. Cryoelectron micrograph analyses and 3D reconstructions showed that the virus retains its overall icosahedral structure at this mildly acidic pH, except in the membrane-binding region, where monomeric E1 associates with the target membrane and the E2 glycoprotein retains its original trimeric organization. The remaining E2 trimers may hinder E1 homotrimerization and are a potential target for antiviral drugs.
History
DepositionMay 14, 2013-
Header (metadata) releaseMay 29, 2013-
Map releaseAug 7, 2013-
UpdateDec 11, 2013-
Current statusDec 11, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 14.9
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 14.9
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image2374.png

Downloads & links

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Map

FileDownload / File: emd_2374.map.gz / Format: CCP4 / Size: 61.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of Sindbis virus when a liposome associates to its 50-fold axis. 5-fold symmetry was applied to the reconstruction map.
Voxel sizeX=Y=Z: 3.951 Å
Density
Contour LevelBy AUTHOR: 14.9 / Movie #1: 14.9
Minimum - Maximum-41.457096100000001 - 59.282108309999998
Average (Standard dev.)1.07148087 (±6.93235254)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-127-127-127
Dimensions255255255
Spacing255255255
CellA=B=C: 1007.505 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.9513.9513.951
M x/y/z255255255
origin x/y/z0.0000.0000.000
length x/y/z1007.5051007.5051007.505
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-127-127-127
NC/NR/NS255255255
D min/max/mean-41.45759.2821.071

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Supplemental data

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Sample components

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Entire : Sindbis virus - liposome complex at pH 6.4

EntireName: Sindbis virus - liposome complex at pH 6.4
Components
  • Sample: Sindbis virus - liposome complex at pH 6.4
  • Virus: Sindbis virus

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Supramolecule #1000: Sindbis virus - liposome complex at pH 6.4

SupramoleculeName: Sindbis virus - liposome complex at pH 6.4 / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Sindbis virus

SupramoleculeName: Sindbis virus / type: virus / ID: 1
Details: The virus was mixed with liposome and was acidified to pH 6.4 at 37C for 2 min
NCBI-ID: 11034 / Sci species name: Sindbis virus / Sci species strain: TE12 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Host systemOrganism: Mesocricetus auratus (golden hamster) / Recombinant cell: BHK-21
Virus shellShell ID: 1 / Name: glycoprotein / Diameter: 700 Å / T number (triangulation number): 4

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6.4
Details: Virus and liposome were suspended in TNE (10mM Tris, 200mM NaCl, 1mM EDTA, pH 7.4). The low pH buffer is (50mM MES, 200mM NaCl, pH.6.4)
GridDetails: Ultra-thin carbon film
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III
Timed resolved state: Vitrified 2 min after waiting in the chamber (37C)

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 75930 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt angle min: 0 / Tilt angle max: 0
TemperatureAverage: 95 K
DateFeb 4, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 25 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: auto3dem / Number images used: 196

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