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- EMDB-5939: Electron cryo-microscopy of the partially glycosylated gp80 form ... -

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Basic information

Entry
Database: EMDB / ID: EMD-5939
TitleElectron cryo-microscopy of the partially glycosylated gp80 form of the wild type furin precursor Env of Moloney murine leukemia virus
Map dataReconstruction of the partially glycosylated form (gp80) of the wild type furin precursor Env of Moloney murine leukemia virus
Sample
  • Sample: Partially glycosylated form (gp80) of wild type furin precursor Env of Moloney murine leukemia virus
  • Protein or peptide: gp80
KeywordsFurin precursor / Moloney murine leukemia virus / Env maturation / gp80
Biological speciesMoloney murine leukemia virus
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 Å
AuthorsSjoberg M / Wu SR / Loving R / Rantalainen K / Lindqvist B / Garoff H
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Furin cleavage of the Moloney murine leukemia virus Env precursor reorganizes the spike structure.
Authors: Mathilda Sjöberg / Shang-Rung Wu / Robin Löving / Kimmo Rantalainen / Birgitta Lindqvist / Henrik Garoff /
Abstract: The trimeric Moloney murine leukemia virus Env protein matures by two proteolytic cleavages. First, furin cleaves the Env precursor into the surface (SU) and transmembrane (TM) subunits in the cell ...The trimeric Moloney murine leukemia virus Env protein matures by two proteolytic cleavages. First, furin cleaves the Env precursor into the surface (SU) and transmembrane (TM) subunits in the cell and then the viral protease cleaves the R-peptide from TM in new virus. Here we analyzed the structure of the furin precursor, by cryoelectron microscopy. We transfected 293T cells with a furin cleavage site provirus mutant, R466G/K468G, and produced the virus in the presence of amprenavir to also inhibit the R-peptide cleavage. Although Env incorporation into particles was inhibited, enough precursor could be isolated and analyzed by cryoelectron microscopy to yield a 3D structure at 22 Å resolution. This showed an open cage-like structure like that of the R-peptide precursor and the mature Env described before. However, the middle protrusion of the protomeric unit, so prominently pointing out from the side of the more mature forms of the Env, was absent. Instead, there was extra density in the top protrusion. This suggested that the C-terminal SU domain was associated alongside the receptor binding N-terminal SU domain in the furin precursor. This was supported by mapping with a SU C-terminal domain-specific antigen binding fragment. We concluded that furin cleavage not only separates the subunits and liberates the fusion peptide at the end of TM but also allows the C-terminal domain to relocate into a peripheral position. This conformational change might explain how the C-terminal domain of SU gains the potential to undergo disulfide isomerization, an event that facilitates membrane fusion.
History
DepositionMar 28, 2014-
Header (metadata) releaseApr 9, 2014-
Map releaseApr 9, 2014-
UpdateMay 14, 2014-
Current statusMay 14, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.85
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.85
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5939.gif

Downloads & links

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Map

FileDownload / File: emd_5939.map.gz / Format: CCP4 / Size: 422.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the partially glycosylated form (gp80) of the wild type furin precursor Env of Moloney murine leukemia virus
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.5 Å/pix.
x 48 pix.
= 168. Å		3.5 Å/pix.
x 48 pix.
= 168. Å		3.5 Å/pix.
x 48 pix.
= 168. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.85 / Movie #1: 2.85
Minimum - Maximum-1.68456912 - 6.63817596
Average (Standard dev.)0.03410213 (±0.90336734)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-7-7-7
Dimensions484848
Spacing484848
CellA=B=C: 168.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.53.53.5
M x/y/z484848
origin x/y/z0.0000.0000.000
length x/y/z168.000168.000168.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-7-7-7
NC/NR/NS484848
D min/max/mean-1.6856.6380.034

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Supplemental data

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Sample components

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Entire : Partially glycosylated form (gp80) of wild type furin precursor E...

EntireName: Partially glycosylated form (gp80) of wild type furin precursor Env of Moloney murine leukemia virus
Components
  • Sample: Partially glycosylated form (gp80) of wild type furin precursor Env of Moloney murine leukemia virus
  • Protein or peptide: gp80

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Supramolecule #1000: Partially glycosylated form (gp80) of wild type furin precursor E...

SupramoleculeName: Partially glycosylated form (gp80) of wild type furin precursor Env of Moloney murine leukemia virus
type: sample / ID: 1000
Details: Affinity purified, gradient separated protein in 0.05% Triton X-100
Oligomeric state: trimer / Number unique components: 1
Molecular weightExperimental: 500 KDa / Theoretical: 270 KDa / Method: Estimated from Blue native PAGE

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Macromolecule #1: gp80

MacromoleculeName: gp80 / type: protein_or_peptide / ID: 1 / Name.synonym: Partially glycosylated furin precursor / Number of copies: 3 / Oligomeric state: Trimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Moloney murine leukemia virus
Molecular weightExperimental: 500 KDa / Theoretical: 270 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.4 / Details: 50 mM HEPES, 100 mM NaCl, 1.8 mM CaCl2, pH 7.4
StainingType: NEGATIVE
Details: The specimen was stained with 2% uranyl acetate (UA).
GridDetails: 400 mesh holey carbon grid, glow discharged
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2100F
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected using online FFT.
DateJan 17, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 3.5 µm / Number real images: 30 / Average electron dose: 9 e/Å2 / Bits/pixel: 14
Electron beamAcceleration voltage: 200 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsCalibrated magnification: 43200 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.072 µm / Nominal defocus min: 2.048 µm / Nominal magnification: 43200
Sample stageSpecimen holder model: OTHER

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Image processing

DetailsThe particles were selected using a semi-automatic selection program in EMAN.
CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: OTHER / Software - Name: EMAN1, EMAN2
Details: Final maps were calculated from a single dataset. The particles were selected using an automatic selection program. Damaged particles were removed by visual inspection.
Number images used: 1329
Final angle assignmentDetails: EMAN
Final two d classificationNumber classes: 20

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