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- EMDB-5040: 3D structure of the ATP-bound yeast Vps4pE233Q dodecamer -

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Basic information

Entry
Database: EMDB / ID: EMD-5040
Title3D structure of the ATP-bound yeast Vps4pE233Q dodecamer
Map data3D density map of ATP-bound yeast Vps4(delta)E233Q
Sample
  • Sample: 6xHis tagged yeast Vps4pE233Q
  • Protein or peptide: Vacuolar protein sorting 4
Keywordsprotein transport / AAA ATPase / endosomal sorting / enveloped virus budding
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsLandsberg MJ / Vajjhala PR / Rothnagel R / Munn AL / Hankamer B
CitationJournal: Structure / Year: 2009
Title: Three-dimensional structure of AAA ATPase Vps4: advancing structural insights into the mechanisms of endosomal sorting and enveloped virus budding.
Authors: Michael John Landsberg / Parimala Rao Vajjhala / Rosalba Rothnagel / Alan Leslie Munn / Ben Hankamer /
Abstract: Vps4 is a AAA ATPase that mediates endosomal membrane protein sorting. It is also a host factor hijacked by a diverse set of clinically important viruses, including HIV and Ebola, to facilitate viral ...Vps4 is a AAA ATPase that mediates endosomal membrane protein sorting. It is also a host factor hijacked by a diverse set of clinically important viruses, including HIV and Ebola, to facilitate viral budding. Here we present the three-dimensional structure of the hydrolysis-defective Vps4p(E233Q) mutant. Single-particle analysis, multiangle laser light scattering, and the docking of independently determined atomic models of Vps4 monomers reveal a complex with C6 point symmetry, distinguishing between a range of previously suggested oligomeric states (8-14 subunits). The 3D reconstruction also reveals a tail-to-tail subunit organization between the two rings of the complex and identifies the location of domains critical to complex assembly and interaction with partner proteins. Our refined Vps4 structure is better supported by independent lines of evidence than those previously proposed, and provides insights into the mechanism of endosomal membrane protein sorting and viral envelope budding.
History
DepositionDec 23, 2008-
Header (metadata) releaseOct 6, 2009-
Map releaseOct 6, 2009-
UpdateOct 6, 2009-
Current statusOct 6, 2009Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 3.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5040_1.png

Downloads & links

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Map

FileDownload / File: emd_5040.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D density map of ATP-bound yeast Vps4(delta)E233Q
Voxel sizeX=Y=Z: 4.087 Å
Density
Contour LevelBy EMDB: 2.5 / Movie #1: 3.2
Minimum - Maximum-8.311529999999999 - 13.5456
Average (Standard dev.)-0.00000000155219 (±0.826126)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-48-48-48
Dimensions969696
Spacing969696
CellA=B=C: 392.352 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.0874.0874.087
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z392.352392.352392.352
α/β/γ90.00090.00090.000
start NX/NY/NZ-20-28-19
NX/NY/NZ415638
MAP C/R/S123
start NC/NR/NS-48-48-48
NC/NR/NS969696
D min/max/mean-8.31213.546-0.000

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Supplemental data

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Sample components

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Entire : 6xHis tagged yeast Vps4pE233Q

EntireName: 6xHis tagged yeast Vps4pE233Q
Components
  • Sample: 6xHis tagged yeast Vps4pE233Q
  • Protein or peptide: Vacuolar protein sorting 4

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Supramolecule #1000: 6xHis tagged yeast Vps4pE233Q

SupramoleculeName: 6xHis tagged yeast Vps4pE233Q / type: sample / ID: 1000 / Oligomeric state: The complex is a dodecamer / Number unique components: 1
Molecular weightExperimental: 560 KDa / Theoretical: 590 KDa / Method: Multi-angle laser light scattering

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Macromolecule #1: Vacuolar protein sorting 4

MacromoleculeName: Vacuolar protein sorting 4 / type: protein_or_peptide / ID: 1 / Name.synonym: Vps4p(E233Q) / Number of copies: 12 / Oligomeric state: Dodecamer / Recombinant expression: Yes
Source (natural)Strain: Saccharomyces cerevisiae / Tissue: cytosol / Location in cell: Endosome membrame
Molecular weightExperimental: 560 KDa / Theoretical: 590 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET11a

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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: 0.1M potassium acetate, 5mM magnesium acetate, 20mM Hepes, 1mM ATP
StainingType: NEGATIVE
Details: Grids with adsorbed protein washed with an aqueous solution of 2% (w/v) uranyl acetate for 30 seconds.
GridDetails: 400 mesh copper grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F30
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 93,000 times magnification
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 170 / Average electron dose: 60 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.1 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 59000
Sample stageSpecimen holder: single tilt / Specimen holder model: OTHER
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

DetailsParticles were selected semi-interactively using SwarmPS
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: IMAGIC, XMIPP, EMAN / Details: FSC at 0.5 cut-off estimates resolution at 18.3 / Number images used: 8590
Final two d classificationNumber classes: 340

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