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- EMDB-4012: Structure of a hexagonal assembly of the PFV glycoprotein from th... -

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Basic information

Entry
Database: EMDB / ID: EMD-4012
TitleStructure of a hexagonal assembly of the PFV glycoprotein from the iFuse Env mutant by cryo-electron microscopy
Map dataNone
Sample
  • Virus: Human spumaretrovirus
Biological speciesHuman spumaretrovirus
Methodsingle particle reconstruction / cryo EM / Resolution: 9.8 Å
AuthorsEffantin G
CitationJournal: PLoS Pathog / Year: 2016
Title: Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture.
Authors: Grégory Effantin / Leandro F Estrozi / Nick Aschman / Patricia Renesto / Nicole Stanke / Dirk Lindemann / Guy Schoehn / Winfried Weissenhorn /
Abstract: Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is ...Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae.
History
DepositionJun 6, 2016-
Header (metadata) releaseJun 22, 2016-
Map releaseJun 22, 2016-
UpdateJul 12, 2017-
Current statusJul 12, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

FileDownload / File: emd_4012.map.gz / Format: CCP4 / Size: 39.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.94 Å
Density
Contour LevelBy ANNOTATOR: 0.017 / Movie #1: 0.015
Minimum - Maximum-0.033737183 - 0.06661329
Average (Standard dev.)0.0006472354 (±0.0062029916)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions218218218
Spacing218218218
CellA=B=C: 422.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.941.941.94
M x/y/z218218218
origin x/y/z0.0000.0000.000
length x/y/z422.920422.920422.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS218218218
D min/max/mean-0.0340.0670.001

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Supplemental data

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Sample components

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Entire : Human spumaretrovirus

EntireName: Human spumaretrovirus
Components
  • Virus: Human spumaretrovirus

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Supramolecule #1: Human spumaretrovirus

SupramoleculeName: Human spumaretrovirus / type: virus / ID: 1 / Parent: 0 / Details: Mutant in the Env polyprotein / NCBI-ID: 11963 / Sci species name: Human spumaretrovirus / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: Yes / Virus empty: No
Host systemOrganism: Homo sapiens (human) / Recombinant plasmid: pcoPG4, pcoPE32, pcoPP
Molecular weightTheoretical: 330 KDa

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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: 7.5
VitrificationCryogen name: ETHANE
DetailsThe iFuse mutant is a variant of Env where the furine cleavage site between the SUrface (gp80 SU ) and TransMembrane (gp48 TM ) domains of Env has been mutated. This results in a partially processed glycoprotein as the cleavage between the LP and SU domains is preserved. Particles are released at nearly wild type level from cells but are non-infectious.

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: OTHER / Details: Initial model is from subtomogram averaging
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 9.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 5543

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