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- EMDB-2066: Electron cryo-microscopy of the L651A mutant R-peptide precursor ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2066
TitleElectron cryo-microscopy of the L651A mutant R-peptide precursor Env of Moloney murine leukemia virus in its native state
Map dataReconstruction of the L651A mutant R-peptide precursor of Moloney murine leukemia virus Env in its native state
Sample
  • Sample: Native form of the L651A mutant R-peptide precursor of Moloney murine leukemia virus Env
  • Protein or peptide: gp70-Pr15E
Keywordscryo-EM / Retrovirus / spike protein / maturation cleavage / R-peptide
Biological speciesMoloney murine leukemia virus
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 22.0 Å
AuthorsLoving R / Wu SR / Sjoberg M / Lindqvist B / Garoff H
CitationJournal: Proc Natl Acad Sci U S A / Year: 2012
Title: Maturation cleavage of the murine leukemia virus Env precursor separates the transmembrane subunits to prime it for receptor triggering.
Authors: Robin Löving / Shang-Rung Wu / Mathilda Sjöberg / Birgitta Lindqvist / Henrik Garoff /
Abstract: The Env protein of murine leukemia virus matures by two cleavage events. First, cellular furin separates the receptor binding surface (SU) subunit from the fusion-active transmembrane (TM) subunit ...The Env protein of murine leukemia virus matures by two cleavage events. First, cellular furin separates the receptor binding surface (SU) subunit from the fusion-active transmembrane (TM) subunit and then, in the newly assembled particle, the viral protease removes a 16-residue peptide, the R-peptide from the endodomain of the TM. Both cleavage events are required to prime the Env for receptor-triggered activation. Cryoelectron microscopy (cryo-EM) analyses have shown that the mature Env forms an open cage-like structure composed of three SU-TM complexes, where the TM subunits formed separated Env legs. Here we have studied the structure of the R-peptide precursor Env by cryo-EM. TM cleavage in Moloney murine leukemia virus was inhibited by amprenavir, and the Envs were solubilized in Triton X-100 and isolated by sedimentation in a sucrose gradient. We found that the legs of the R-peptide Env were held together by trimeric interactions at the very bottom of the Env. This suggested that the R-peptide ties the TM legs together and that this prevents the activation of the TM for fusion. The model was supported by further cryo-EM studies using an R-peptide Env mutant that was fusion-competent despite an uncleaved R-peptide. The Env legs of this mutant were found to be separated, like in the mature Env. This shows that it is the TM leg separation, normally caused by R-peptide cleavage, that primes the Env for receptor triggering.
History
DepositionApr 4, 2012-
Header (metadata) releaseApr 20, 2012-
Map releaseSep 26, 2012-
UpdateSep 26, 2012-
Current statusSep 26, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD2066.png

Downloads & links

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Map

FileDownload / File: emd_2066.map.gz / Format: CCP4 / Size: 422.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the L651A mutant R-peptide precursor of Moloney murine leukemia virus Env in its native state
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: 1.6 / Movie #1: 1.6
Minimum - Maximum-4.73799372 - 5.21405506
Average (Standard dev.)0.01628768 (±0.86803526)
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/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-7-7-7
NC/NR/NS484848
D min/max/mean-4.7385.2140.016

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Supplemental data

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Sample components

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Entire : Native form of the L651A mutant R-peptide precursor of Moloney mu...

EntireName: Native form of the L651A mutant R-peptide precursor of Moloney murine leukemia virus Env
Components
  • Sample: Native form of the L651A mutant R-peptide precursor of Moloney murine leukemia virus Env
  • Protein or peptide: gp70-Pr15E

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Supramolecule #1000: Native form of the L651A mutant R-peptide precursor of Moloney mu...

SupramoleculeName: Native form of the L651A mutant R-peptide precursor of Moloney murine leukemia virus Env
type: sample / ID: 1000 / Oligomeric state: trimeric / Number unique components: 1
Molecular weightExperimental: 500 KDa / Theoretical: 270 KDa / Method: Blue native PAGE

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Macromolecule #1: gp70-Pr15E

MacromoleculeName: gp70-Pr15E / type: protein_or_peptide / ID: 1 / Name.synonym: (SU-TM)3; Env / Details: Expressed in Human Embryonic Kidney 293T cells / Number of copies: 1 / Oligomeric state: trimeric / Recombinant expression: Yes
Source (natural)Organism: Moloney murine leukemia virus / synonym: MO-MLV
Molecular weightExperimental: 500 KDa / Theoretical: 270 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant plasmid: pNCA

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Experimental details

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Structure determination

Methodnegative staining, cryo 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, 0.05% Triton X-100
StainingType: NEGATIVE
Details: The specimen was frozen in liquid ethane and transferred to liquid nitrogen for EM inspection without staining.
GridDetails: 400 mesh holey carbon grid. The grids were glow discharged.
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK II
Timed resolved state: Vitrified 45 msec after spraying with effector
Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeJEOL 2100F
TemperatureMin: 93 K / Max: 96 K / Average: 95 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected using online FFT
DateJul 1, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC CCD / Digitization - Sampling interval: 3.5 µm / Number real images: 653 / Average electron dose: 9 e/Å2 / Bits/pixel: 14
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 43200 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 43200
Sample stageSpecimen holder: liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsThe particles were selected using an automatic selection program and the damaged particles were removed by visual inspection.
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN1, EMAN2
Details: Final maps were calculated from four averaged datasets
Number images used: 4702
Final two d classificationNumber classes: 131

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