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- EMDB-1358: Atypical AAA+ subunit packing creates an expanded cavity for disa... -

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Basic information

Entry
Database: EMDB / ID: EMD-1358
TitleAtypical AAA+ subunit packing creates an expanded cavity for disaggregation by the protein-remodeling factor Hsp104.
Map dataYeast Hsp104 N728A 3D density map. ATPgammaS bound hexamer.
Sample
  • Sample: Yeast Hsp104 N728A ATPgS
  • Protein or peptide: Hsp104 N728A
  • Ligand: ATPgammaS
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.0 Å
AuthorsWendler P / Shorter J / Plisson C / Cashikar AG / Lindquist S / Saibil HR
CitationJournal: Cell / Year: 2007
Title: Atypical AAA+ subunit packing creates an expanded cavity for disaggregation by the protein-remodeling factor Hsp104.
Authors: Petra Wendler / James Shorter / Celia Plisson / Anil G Cashikar / Susan Lindquist / Helen R Saibil /
Abstract: Hsp104, a yeast protein-remodeling factor of the AAA+ (ATPases associated with various cellular activities) superfamily, and its homologs in bacteria and plants mediate cell recovery after severe ...Hsp104, a yeast protein-remodeling factor of the AAA+ (ATPases associated with various cellular activities) superfamily, and its homologs in bacteria and plants mediate cell recovery after severe stress by disaggregating denatured proteins through a poorly understood mechanism. Here, we present cryo-electron microscopy maps and domain fitting of Hsp104 hexamers, revealing an unusual arrangement of AAA+ modules with the prominent coiled-coil domain intercalated between the AAA+ domains. This packing results in a greatly expanded cavity, which is capped at either end by N- and C-terminal domains. The fitted structures as well as mutation of conserved coiled-coil arginines suggest that the coiled-coil domain plays a major role in the extraction of proteins from aggregates, providing conserved residues for key functions in ATP hydrolysis and potentially for substrate interaction. The large cavity could enable the uptake of polypeptide loops without a requirement for exposed N or C termini.
History
DepositionMay 16, 2007-
Header (metadata) releaseMay 16, 2007-
Map releaseJan 3, 2008-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1358.gif

Downloads & links

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Map

FileDownload / File: emd_1358.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast Hsp104 N728A 3D density map. ATPgammaS bound hexamer.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 128 pix.
= 358.4 Å		2.8 Å/pix.
x 128 pix.
= 358.4 Å		2.8 Å/pix.
x 128 pix.
= 358.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.57 / Movie #1: 0.5
Minimum - Maximum-1.37277 - 9.965070000000001
Average (Standard dev.)0.0346584 (±0.356831)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-63-64
Dimensions128128128
Spacing128128128
CellA=B=C: 358.4 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z358.400358.400358.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-63-64-64
NC/NR/NS128128128
D min/max/mean-1.3739.9650.035

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Supplemental data

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Sample components

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Entire : Yeast Hsp104 N728A ATPgS

EntireName: Yeast Hsp104 N728A ATPgS
Components
  • Sample: Yeast Hsp104 N728A ATPgS
  • Protein or peptide: Hsp104 N728A
  • Ligand: ATPgammaS

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Supramolecule #1000: Yeast Hsp104 N728A ATPgS

SupramoleculeName: Yeast Hsp104 N728A ATPgS / type: sample / ID: 1000 / Oligomeric state: hexamer / Number unique components: 2
Molecular weightExperimental: 600 KDa / Theoretical: 612 KDa / Method: Gel filtration Glutaraldehyde cross-linking

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Macromolecule #1: Hsp104 N728A

MacromoleculeName: Hsp104 N728A / type: protein_or_peptide / ID: 1 / Name.synonym: Heat Shock Protein 104 / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast / Location in cell: cytoplasm, nucleus
Molecular weightExperimental: 102 KDa / Theoretical: 100 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pNOTAG

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Macromolecule #2: ATPgammaS

MacromoleculeName: ATPgammaS / type: ligand / ID: 2 / Recombinant expression: No

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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

Concentration0.3 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES pH 7.5, 20 mM NaCl, 10 mM MgCl2, 1 mM DTT, 2mM ATPgS
GridDetails: 300 mesh copper grid- holey carbon film
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: home made
Method: The grids were blotted for 2-3 sec and immediately plunged into liquid ethane

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 77 K / Max: 85 K / Average: 77 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected for at
DateMar 17, 2005
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 35 / Average electron dose: 15 e/Å2 / Od range: 1 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.9 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 50000
Sample stageSpecimen holder: single tilt cryo / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: phase flipping, each particle
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Imagic, Spider / Number images used: 7211

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Atomic model buiding 1

DetailsThe domains were manually fitted as rigid bodies using Pymol. Automated fitting in Chimera optimised fit for NBD2.
RefinementProtocol: RIGID BODY FIT

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