EMDB-4007: Structure of iFuse PFV glycoprotein by cryo-electron tomography

Basic information

• Entry: Database: EMDB / ID: EMD-4007
• Title: Structure of iFuse PFV glycoprotein by cryo-electron tomography
• Map data: PFV iFuse Glycoprotein

Sample

• Virus: Human spumaretrovirus

• Biological species: Human spumaretrovirus
• Method: subtomogram averaging / cryo EM / Resolution: 28.0 Å
• Authors: Effantin G
• Citation: Journal: 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 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

Deposition	Jun 3, 2016	-
Header (metadata) release	Jun 22, 2016	-
Map release	Jun 22, 2016	-
Update	Jul 20, 2016	-
Current status	Jul 20, 2016	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_4007.map.gz / Format: CCP4 / Size: 1.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: PFV iFuse Glycoprotein
• Voxel size: X=Y=Z: 7.6 Å

Density

Contour Level	By AUTHOR: 0.145 / Movie #1: 0.145
Minimum - Maximum	-0.5976977 - 0.714458
Average (Standard dev.)	0.0063638696 (±0.19410585)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin -35 -35 -35 Dimensions 70 70 70 Spacing 70 70 70
Cell	A=B=C: 532.0 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	7.6	7.6	7.6
M x/y/z	70	70	70
origin x/y/z	0.000	0.000	0.000
length x/y/z	532.000	532.000	532.000
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-147	-147	-146
NX/NY/NZ	294	294	294
MAP C/R/S	1	2	3
start NC/NR/NS	-35	-35	-35
NC/NR/NS	70	70	70
D min/max/mean	-0.598	0.714	0.006

Supplemental data

Sample components

Entire : Human spumaretrovirus

• Entire: Name: Human spumaretrovirus

Components

• Virus: Human spumaretrovirus

Supramolecule #1: Human spumaretrovirus

• Supramolecule: Name: Human spumaretrovirus / type: virus / ID: 1 / Parent: 0 / Details: Mutant in the Env glycoprotein / NCBI-ID: 11963 / Sci species name: Human spumaretrovirus / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: Yes / Virus empty: No
• Host system: Organism: Homo sapiens (human) / Recombinant plasmid: pcoPG4, pcoPE32, pcoPP
• Molecular weight: Theoretical: 330 KDa

Experimental details

Structure determination

• Method: cryo EM
• Processing: subtomogram averaging
• Aggregation state: particle

Sample preparation

• Buffer: pH: 7.5
• Vitrification: Cryogen name: ETHANE
• Details: The 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

Electron microscopy

• Microscope: FEI TECNAI F20
• Image recording: Film or detector model: FEI EAGLE (4k x 4k) / Average electron dose: 1.0 e/Ų
• Electron beam: Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
• Experimental equipment: Model: Tecnai F20 / Image courtesy: FEI Company

Image processing

• Final reconstruction: Applied symmetry - Point group: C₃ (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 28.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PEET / Number subtomograms used: 3000
• Extraction: Number tomograms: 7 / Number images used: 3930
• Final angle assignment: Type: PROJECTION MATCHING