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- EMDB-1601: Motor mechanism for protein threading through Hsp104 -

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Basic information

Entry
Database: EMDB / ID: EMD-1601
TitleMotor mechanism for protein threading through Hsp104
Map dataYeast Hsp104 N728A 3D density map. Hexamer formed in the presence of ATP. Reconstruction without imposed symmetry.
Sample
  • Sample: S. cerevisiae Hsp104 N728A ATP, asymmetric reconstruction
  • Protein or peptide: Hsp104 N728A
KeywordsClp/Hsp100 / AAA+ / protein remodeling / disaggregation / molecular machines / heat shock protein / Hsp104 / ClpB / yeast prions
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.1 Å
AuthorsWendler P / Shorter J / Snead D / Plisson C / Clare DK / Lindquist S / Saibil H
CitationJournal: Mol Cell / Year: 2009
Title: Motor mechanism for protein threading through Hsp104.
Authors: Petra Wendler / James Shorter / David Snead / Celia Plisson / Daniel K Clare / Susan Lindquist / Helen R Saibil /
Abstract: The protein-remodeling machine Hsp104 dissolves amorphous aggregates as well as ordered amyloid assemblies such as yeast prions. Force generation originates from a tandem AAA+ (ATPases associated ...The protein-remodeling machine Hsp104 dissolves amorphous aggregates as well as ordered amyloid assemblies such as yeast prions. Force generation originates from a tandem AAA+ (ATPases associated with various cellular activities) cassette, but the mechanism and allostery of this action remain to be established. Our cryoelectron microscopy maps of Hsp104 hexamers reveal substantial domain movements upon ATP binding and hydrolysis in the first nucleotide-binding domain (NBD1). Fitting atomic models of Hsp104 domains to the EM density maps plus supporting biochemical measurements show how the domain movements displace sites bearing the substrate-binding tyrosine loops. This provides the structural basis for N- to C-terminal substrate threading through the central cavity, enabling a clockwise handover of substrate in the NBD1 ring and coordinated substrate binding between NBD1 and NBD2. Asymmetric reconstructions of Hsp104 in the presence of ATPgammaS or ATP support sequential rather than concerted ATP hydrolysis in the NBD1 ring.
History
DepositionFeb 10, 2009-
Header (metadata) releaseFeb 18, 2009-
Map releaseApr 21, 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: 0.12
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1601.png

Downloads & links

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Map

FileDownload / File: emd_1601.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast Hsp104 N728A 3D density map. Hexamer formed in the presence of ATP. Reconstruction without imposed symmetry.
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 LevelBy AUTHOR: 0.1 / Movie #1: 0.12
Minimum - Maximum-1.99803 - 10.0
Average (Standard dev.)0.025259 (±0.317221)
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-17-17-200
NX/NY/NZ123123401
MAP C/R/S123
start NC/NR/NS-63-64-64
NC/NR/NS128128128
D min/max/mean-1.99810.0000.025

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Supplemental data

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Sample components

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Entire : S. cerevisiae Hsp104 N728A ATP, asymmetric reconstruction

EntireName: S. cerevisiae Hsp104 N728A ATP, asymmetric reconstruction
Components
  • Sample: S. cerevisiae Hsp104 N728A ATP, asymmetric reconstruction
  • Protein or peptide: Hsp104 N728A

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Supramolecule #1000: S. cerevisiae Hsp104 N728A ATP, asymmetric reconstruction

SupramoleculeName: S. cerevisiae Hsp104 N728A ATP, asymmetric reconstruction
type: sample / ID: 1000 / Oligomeric state: hexamer / Number unique components: 2
Molecular weightTheoretical: 612 KDa

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Macromolecule #1: Hsp104 N728A

MacromoleculeName: Hsp104 N728A / type: protein_or_peptide / ID: 1 / Name.synonym: Hsp104 N728A / Details: Hexamer formation in the presence of 2-5 mM ATP / Number of copies: 6 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm, nucleus
Molecular weightTheoretical: 102 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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, 2-5mM ATP
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: corrected for at specimen level
DateJan 28, 2004
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 43 / 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: 3.5 µm / Nominal defocus min: 1.5 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: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.1 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC SPIDER MRC / Details: asymmetric reconstruction / Number images used: 11128

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