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- EMDB-1481: cryo electron microscopy structure of Vps4p AMPPNP-complexed tetr... -

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Basic information

Entry
Database: EMDB / ID: EMD-1481
Titlecryo electron microscopy structure of Vps4p AMPPNP-complexed tetradecamer
Map dataThis is a 3D reconstruction of a Vps4p AMPPNP-complexed tetradecamer. The N-terminal MIT-domain is deleted.
Sample
  • Sample: His-tagged Vps4p AAA-ATPase cassette
  • Protein or peptide: Vps4p
Keywordsvacuolar protein sorting / AAA-ATPase / ESCRT / HIV / Vps
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 18.0 Å
AuthorsHartmann C / Chami M / Zachariae U / de Groot BL / Engel A / Gruetter MG
CitationJournal: J Mol Biol / Year: 2008
Title: Vacuolar protein sorting: two different functional states of the AAA-ATPase Vps4p.
Authors: Claudia Hartmann / Mohamed Chami / Ulrich Zachariae / Bert L de Groot / Andreas Engel / Markus G Grütter /
Abstract: The vacuolar protein sorting (Vps) pathway, in which Vps4 class I AAA-ATPases play a central role, regulates growth factor receptors, immune response, and developmental signaling, and participates in ...The vacuolar protein sorting (Vps) pathway, in which Vps4 class I AAA-ATPases play a central role, regulates growth factor receptors, immune response, and developmental signaling, and participates in tumor suppression, apoptosis, and retrovirus budding. We present the first atomic structure of the nucleotide-free yeast His(6)DeltaNVps4p dimer and its AMPPNP (5'-adenylyl-beta,gamma-imidodiphosphate)-bound tetradecamer, derived from a cryo electron microscopy map. Vps4p dimers form two distinct heptameric rings and accommodate AAA cassettes in a head-to-head--not in a head-to-tail-fashion as in class II AAA-ATPases. Our model suggests a mechanism for disassembling ESCRT (endosomal sorting complex required for transport) complexes by movements of substrate-binding domains located at the periphery of the tetradecamer during ATP hydrolysis in one ring, followed by translocation through the central pore and ATP hydrolysis in the second ring.
History
DepositionFeb 25, 2008-
Header (metadata) releaseFeb 25, 2008-
Map releaseMar 31, 2009-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.23965049
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.23965049
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1481.gif

Downloads & links

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Map

FileDownload / File: emd_1481.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a 3D reconstruction of a Vps4p AMPPNP-complexed tetradecamer. The N-terminal MIT-domain is deleted.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 128 pix.
= 254. Å		2 Å/pix.
x 128 pix.
= 254. Å		2 Å/pix.
x 128 pix.
= 254. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 1.3 / Movie #1: 1.2396505
Minimum - Maximum-9.8514 - 4.5794
Average (Standard dev.)0.00000000925385 (±0.735361)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 254 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z127127127
origin x/y/z0.0000.0000.000
length x/y/z254.000254.000254.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-55-55-55
NX/NY/NZ111111111
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-9.8514.5790.000

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Supplemental data

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Sample components

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Entire : His-tagged Vps4p AAA-ATPase cassette

EntireName: His-tagged Vps4p AAA-ATPase cassette
Components
  • Sample: His-tagged Vps4p AAA-ATPase cassette
  • Protein or peptide: Vps4p

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Supramolecule #1000: His-tagged Vps4p AAA-ATPase cassette

SupramoleculeName: His-tagged Vps4p AAA-ATPase cassette / type: sample / ID: 1000 / Details: fresh prepared / Oligomeric state: tetradecamer / Number unique components: 1
Molecular weightExperimental: 512 KDa / Theoretical: 512 KDa / Method: cryo electron microscopy

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Macromolecule #1: Vps4p

MacromoleculeName: Vps4p / type: protein_or_peptide / ID: 1 / Name.synonym: Vps4p / Number of copies: 14 / Oligomeric state: tetradecamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: bakers' yeast / Location in cell: cytosol
Molecular weightExperimental: 512 KDa / Theoretical: 512 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pet20b

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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

Concentration2.5 mg/mL
BufferpH: 7.5 / Details: 20 mM Tris
GridDetails: holey carbon film
VitrificationCryogen name: ETHANE / Chamber temperature: 97 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: home made Guillotine. add AMPPNP 15min before freezing. Use the Quantifoil holey carbone grids.
Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Cryholder Gatan 626, / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 97 K / Max: 97 K / Average: 97 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism corrected at 50 000
Legacy - Electron beam tilt params: no tilt
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 10 µm / Number real images: 80 / Average electron dose: 10 e/Å2 / Bits/pixel: 8

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Image processing

CTF correctionDetails: ctftilt, SPIDER
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 2473
Detailsboxer

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