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- EMDB-1536: ESCRT-III subunits CHMP2A and CHMP3 assemble in vitro into helica... -

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Basic information

Entry
Database: EMDB / ID: EMD-1536
TitleESCRT-III subunits CHMP2A and CHMP3 assemble in vitro into helical tubular structures
Map dataESCRT-III subunits CHMP2A and CHMP3 assemble in vitro into helical tubular structures
Sample
  • Sample: CHMP proteins
  • Protein or peptide: CHMP
Keywordshelical reconstruction
Biological speciesSaccharomyces (fungus)
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 32.0 Å
AuthorsLata S / Schoehn G / Jain A / Pires R / Piehler J / Goettlinger H / Weissenhorn W
CitationJournal: Science / Year: 2008
Title: Helical structures of ESCRT-III are disassembled by VPS4.
Authors: Suman Lata / Guy Schoehn / Ankur Jain / Ricardo Pires / Jacob Piehler / Heinrich G Gottlinger / Winfried Weissenhorn /
Abstract: During intracellular membrane trafficking and remodeling, protein complexes known as the ESCRTs (endosomal sorting complexes required for transport) interact with membranes and are required for ...During intracellular membrane trafficking and remodeling, protein complexes known as the ESCRTs (endosomal sorting complexes required for transport) interact with membranes and are required for budding processes directed away from the cytosol, including the budding of intralumenal vesicles to form multivesicular bodies; for the budding of some enveloped viruses; and for daughter cell scission in cytokinesis. We found that the ESCRT-III proteins CHMP2A and CHMP3 (charged multivesicular body proteins 2A and 3) could assemble in vitro into helical tubular structures that expose their membrane interaction sites on the outside of the tubule, whereas the AAA-type adenosine triphosphatase VPS4 could bind on the inside of the tubule and disassemble the tubes upon adenosine triphosphate hydrolysis. CHMP2A and CHMP3 copolymerized in solution, and their membrane targeting was cooperatively enhanced on planar lipid bilayers. Such helical CHMP structures could thus assemble within the neck of an inwardly budding vesicle, catalyzing late steps in budding under the control of VPS4.
History
DepositionJul 21, 2008-
Header (metadata) releaseJul 22, 2008-
Map releaseNov 5, 2010-
UpdateNov 9, 2010-
Current statusNov 9, 2010Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0017
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0017
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1536.png

Downloads & links

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Map

FileDownload / File: emd_1536.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationESCRT-III subunits CHMP2A and CHMP3 assemble in vitro into helical tubular structures
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesY (Sec.)X (Row.)Z (Col.)
3.5 Å/pix.
x 240 pix.
= 840. Å		3.5 Å/pix.
x 240 pix.
= 840. Å		3.5 Å/pix.
x 240 pix.
= 840. 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 EMDB: 0.0017 / Movie #1: 0.0017
Minimum - Maximum0.0 - 0.00327867
Average (Standard dev.)0.000164445 (±0.000481635)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZXY
Origin-120-120-120
Dimensions240240240
Spacing240240240
CellA=B=C: 840 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.53.53.5
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z840.000840.000840.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-120-120-120
NX/NY/NZ240240240
MAP C/R/S312
start NC/NR/NS-120-120-120
NC/NR/NS240240240
D min/max/mean0.0000.0030.000

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Supplemental data

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Sample components

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Entire : CHMP proteins

EntireName: CHMP proteins
Components
  • Sample: CHMP proteins
  • Protein or peptide: CHMP

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Supramolecule #1000: CHMP proteins

SupramoleculeName: CHMP proteins / type: sample / ID: 1000 / Oligomeric state: oligomer of dimers / Number unique components: 1

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Macromolecule #1: CHMP

MacromoleculeName: CHMP / type: protein_or_peptide / ID: 1 / Name.synonym: CHMP
Details: CHMP2AdeltaC (9-161)-CHMP3deltaC(9 to 183) . MBP attached to CHMP2AdeltaC
Oligomeric state: oligomer of dimers / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces (fungus) / synonym: Yeast / Cell: E coli
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pETM40

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.6 / Details: 20 mM HEPES, pH 7.6, 150 mM NaCl
StainingType: NEGATIVE
Details: Vitrified samples were prepared according to the method of Dubochet et al.,
GridDetails: Quantifoil R2/2
VitrificationCryogen name: ETHANE / Chamber temperature: 100 K / Instrument: OTHER / Details: Vitrification instrument: Zeiss / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM200T
TemperatureAverage: 100 K
Alignment procedureLegacy - Astigmatism: orrected at 100,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 10 / Average electron dose: 10 e/Å2 / Details: Images binned after scanning / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 39500 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 38000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsParticles were selected by hand using X3d
Final reconstructionApplied symmetry - Helical parameters - Δz: 32 Å
Applied symmetry - Helical parameters - Δ&Phi: 21.71 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 32.0 Å / Resolution method: OTHER / Software - Name: IHRSR / Details: 2500 out of 4400 particles have been used
CTF correctionDetails: each particle

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