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- EMDB-5938: Electron cryo-microscopy of the Moloney murine leukemia virus fur... -

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Entry
Database: EMDB / ID: EMD-5938
TitleElectron cryo-microscopy of the Moloney murine leukemia virus furin precursor Env in its native form in complex with 83A25 Fab
Map dataReconstruction of the native furin cleavage deficient mutant (R466G/K468G) Env of Moloney murine leukemia virus in complex with the 83A25 Fab
Sample
  • Sample: Native form of furin cleavage deficient mutant (R466G/K468G) Env of Moloney murine leukemia virus in complex with 83A25 Fab
  • Protein or peptide: gp90
  • Protein or peptide: monoclonal antibody 83A25 Fab
KeywordsFurin precursor / Moloney murine leukemia virus / Env maturation / 83A25 Fab
Biological speciesMoloney murine leukemia virus / Rattus (rat)
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: 7.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 7.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5938.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the native furin cleavage deficient mutant (R466G/K468G) Env of Moloney murine leukemia virus in complex with the 83A25 Fab
Voxel sizeX=Y=Z: 3.5 Å
Density
Contour LevelBy AUTHOR: 7.4 / Movie #1: 7.4
Minimum - Maximum-3.59785509 - 13.54157925
Average (Standard dev.)0.06688309 (±1.77408254)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 224.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.53.53.5
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z224.000224.000224.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-3.59813.5420.067

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Supplemental data

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Sample components

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Entire : Native form of furin cleavage deficient mutant (R466G/K468G) Env ...

EntireName: Native form of furin cleavage deficient mutant (R466G/K468G) Env of Moloney murine leukemia virus in complex with 83A25 Fab
Components
  • Sample: Native form of furin cleavage deficient mutant (R466G/K468G) Env of Moloney murine leukemia virus in complex with 83A25 Fab
  • Protein or peptide: gp90
  • Protein or peptide: monoclonal antibody 83A25 Fab

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Supramolecule #1000: Native form of furin cleavage deficient mutant (R466G/K468G) Env ...

SupramoleculeName: Native form of furin cleavage deficient mutant (R466G/K468G) Env of Moloney murine leukemia virus in complex with 83A25 Fab
type: sample / ID: 1000
Details: Affinity purified, gradient separated protein in 0.05% Triton X-100
Oligomeric state: trimer / Number unique components: 2
Molecular weightExperimental: 600 KDa / Theoretical: 400 KDa / Method: Estimated from Blue native PAGE

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Macromolecule #1: gp90

MacromoleculeName: gp90 / type: protein_or_peptide / ID: 1 / Name.synonym: 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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Macromolecule #2: monoclonal antibody 83A25 Fab

MacromoleculeName: monoclonal antibody 83A25 Fab / type: protein_or_peptide / ID: 2 / Details: 2-3 Fab molecules bind to the gp90 trimer. / Number of copies: 3 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Rattus (rat) / synonym: rat
Molecular weightExperimental: 50 KDa / Theoretical: 50 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 regular continuous carbon grid, glow discharged
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2100F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 43200 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.072 µm / Nominal defocus min: 2.048 µm / Nominal magnification: 43200
Sample stageSpecimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected using online FFT.
DateJul 5, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 3.5 µm / Number real images: 60 / Average electron dose: 9 e/Å2 / Bits/pixel: 14

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 20
Final angle assignmentDetails: EMAN
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: OTHER / Software - Name: EMAN1, EMAN2
Details: Final maps were calculated from three averaged datasets. The particles were selected using an automatic selection program. Damaged particles were removed by visual inspection.
Number images used: 9374
DetailsThe particles were selected using a semi-automatic selection program in EMAN.

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: Chimera
DetailsAtomic structure of the Fv fragment of the monoclonal Ab 7E2 against the Paracoccus denitrificans cytochrome c oxidase was fitted into the density map.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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