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- EMDB-5937: Electron cryo-microscopy of the Moloney murine leukemia virus fur... -

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Basic information

Entry
Database: EMDB / ID: EMD-5937
TitleElectron cryo-microscopy of the Moloney murine leukemia virus furin precursor Env in its isomerization arrested state (IAS) form
Map dataReconstruction of the furin precursor of the Moloney murine leukemia virus at isomerization arrested intermediate stage (IAS)
Sample
  • Sample: Isomerization arrested stage (IAS) of the furin cleavage deficient mutant (R466G/K468G) Env of Moloney murine leukemia virus
  • Protein or peptide: gp90
KeywordsFurin precursor / Moloney murine leukemia virus / Env maturation / intermediate form
Biological speciesMoloney murine leukemia virus
Methodsingle particle reconstruction / cryo EM / Resolution: 23.0 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: 2.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5937.gif

Downloads & links

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Map

FileDownload / File: emd_5937.map.gz / Format: CCP4 / Size: 422.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the furin precursor of the Moloney murine leukemia virus at isomerization arrested intermediate stage (IAS)
Voxel sizeX=Y=Z: 3.5 Å
Density
Contour LevelBy AUTHOR: 2.6 / Movie #1: 2.6
Minimum - Maximum-3.14429808 - 6.27997875
Average (Standard dev.)0.02771065 (±0.87513155)
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-3.1446.2800.028

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Supplemental data

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Sample components

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Entire : Isomerization arrested stage (IAS) of the furin cleavage deficien...

EntireName: Isomerization arrested stage (IAS) of the furin cleavage deficient mutant (R466G/K468G) Env of Moloney murine leukemia virus
Components
  • Sample: Isomerization arrested stage (IAS) of the furin cleavage deficient mutant (R466G/K468G) Env of Moloney murine leukemia virus
  • Protein or peptide: gp90

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Supramolecule #1000: Isomerization arrested stage (IAS) of the furin cleavage deficien...

SupramoleculeName: Isomerization arrested stage (IAS) of the furin cleavage deficient mutant (R466G/K468G) 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: 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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Experimental details

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Structure determination

Methodcryo EM
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
GridDetails: 400 mesh holey carbon grid, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK II
Method: Blotted for 3 seconds before plunging in liquid ethane, followed by transfer into liquid nitrogen.

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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: 4.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 43200
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 93 K / Max: 96 K / Average: 95 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected using online FFT.
DateJan 25, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 3.5 µm / Number real images: 456 / Average electron dose: 9 e/Å2 / Bits/pixel: 14

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 30
Final angle assignmentDetails: EMAN
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: EMAN1, EMAN2
Details: Final maps were calculated from seven averaged datasets. The particles were selected using an automatic selection program. Damaged particles were removed after visual inspection.
Number images used: 3825
DetailsThe particles were selected using a semi-automatic selection program in EMAN.

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Atomic model buiding 1

Initial modelPDB ID:

1aol

SoftwareName: Chimera
DetailsThe atomic model for the Mo-RBD was obtained using the atomic structure of the highly homologous F-RBD (Fass et al, 1997) (PDB ID 1AOL) and the SWISS-MODEL protein structure homology-modeling server (accessible through the ExPASy web server).
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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