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- EMDB-2151: Electron cryo-microscopy of escrt-III helical polymer -

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Basic information

Entry
Database: EMDB / ID: EMD-2151
TitleElectron cryo-microscopy of escrt-III helical polymer
Map dataReconstruction of chmp2a-3 tubes
Sample
  • Sample: ESCRT-III Chmp2a - Chmp3 co-polymer
  • Protein or peptide: chmp2a
  • Protein or peptide: chmp3
Keywordsvirus budding / membrane deformation
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 22.4 Å
AuthorsEffantin G / Dordor A / Sandrin V / Martinelli N / Sundquist WI / Schoehn G / Weissenhorn W
CitationJournal: Cell Microbiol / Year: 2013
Title: ESCRT-III CHMP2A and CHMP3 form variable helical polymers in vitro and act synergistically during HIV-1 budding.
Authors: Grégory Effantin / Aurélien Dordor / Virginie Sandrin / Nicolas Martinelli / Wesley I Sundquist / Guy Schoehn / Winfried Weissenhorn /
Abstract: The endosomal sorting complex required for transport-III (ESCRT-III) proteins are essential for budding of some enveloped viruses, for the formation of intraluminal vesicles at the endosome and for ...The endosomal sorting complex required for transport-III (ESCRT-III) proteins are essential for budding of some enveloped viruses, for the formation of intraluminal vesicles at the endosome and for the abscission step of cytokinesis. ESCRT-III proteins form polymers that constrict membrane tubes, leading to fission. We have used electron cryomicroscopy to determine the molecular organization of pleiomorphic ESCRT-III CHMP2A-CHMP3 polymers. The three-dimensional reconstruction at 22 Å resolution reveals a helical organization of filaments of CHMP molecules organized in a head-to-tail fashion. Protease susceptibility experiments indicate that polymerization is achieved via conformational changes that increase the protomer stability. Combinatorial siRNA knockdown experiments indicate that CHMP3 contributes synergistically to HIV-1 budding, and the CHMP3 contribution is ~ 10-fold more pronounced in concert with CHMP2A than with CHMP2B. This is consistent with surface plasmon resonance affinity measurements that suggest sequential CHMP4B-CHMP3-CHMP2A recruitment while showing that both CHMP2A and CHMP2B interact with CHMP4B, in agreement with their redundant functions in HIV-1 budding. Our data thus indicate that the CHMP2A-CHMP3 polymer observed in vitro contributes to HIV-1 budding by assembling on CHMP4B polymers.
History
DepositionJun 27, 2012-
Header (metadata) releaseOct 3, 2012-
Map releaseOct 24, 2012-
UpdateApr 20, 2016-
Current statusApr 20, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.4
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image2151.jpg

Downloads & links

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Map

FileDownload / File: emd_2151.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of chmp2a-3 tubes
Voxel sizeX=Y=Z: 3.59 Å
Density
Contour LevelBy AUTHOR: 1.4 / Movie #1: 1.4
Minimum - Maximum-3.93818498 - 5.16021681
Average (Standard dev.)0.02379481 (±0.92937845)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 718.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.593.593.59
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z718.000718.000718.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ454586
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-3.9385.1600.024

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Supplemental data

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Sample components

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Entire : ESCRT-III Chmp2a - Chmp3 co-polymer

EntireName: ESCRT-III Chmp2a - Chmp3 co-polymer
Components
  • Sample: ESCRT-III Chmp2a - Chmp3 co-polymer
  • Protein or peptide: chmp2a
  • Protein or peptide: chmp3

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Supramolecule #1000: ESCRT-III Chmp2a - Chmp3 co-polymer

SupramoleculeName: ESCRT-III Chmp2a - Chmp3 co-polymer / type: sample / ID: 1000 / Number unique components: 2

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Macromolecule #1: chmp2a

MacromoleculeName: chmp2a / type: protein_or_peptide / ID: 1
Details: chmp2a mbp tag was cleaved by tev protease before imaging
Recombinant expression: Yes / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: human / Location in cell: plasma membrane

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Macromolecule #2: chmp3

MacromoleculeName: chmp3 / type: protein_or_peptide / ID: 2 / Recombinant expression: Yes / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: human / Location in cell: plasma membrane

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.6 / Details: 20 mM Hepes pH 7.6, 150 mM NaCl
GridDetails: 400 mesh copper quantifoil grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.3 mm / Nominal magnification: 39000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateOct 10, 2010
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 78 / Average electron dose: 15 e/Å2 / Bits/pixel: 8
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Helical parameters - Δz: 6.03 Å
Applied symmetry - Helical parameters - Δ&Phi: 11.9 °
Applied symmetry - Helical parameters - Axial symmetry: C6 (6 fold cyclic)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.4 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: ctffind, bsof, spider, imagic
DetailsThe particles were aligned using IHRSR

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