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- EMDB-1631: 3D EM reconstruction of Hsp104(E687A,E285A) ATP bound state -

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Basic information

Entry
Database: EMDB / ID: EMD-1631
Title3D EM reconstruction of Hsp104(E687A,E285A) ATP bound state
Map dataHsp104(E285A,E687A) Trap mutant ATP bound state
Sample
  • Sample: Hsp104
  • Protein or peptide: Hsp104
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.0 Å
AuthorsLee S / Sielaff B / Lee J / Tsai FTF
CitationJournal: Proc Natl Acad Sci U S A / Year: 2010
Title: CryoEM structure of Hsp104 and its mechanistic implication for protein disaggregation.
Authors: Sukyeong Lee / Bernhard Sielaff / Jungsoon Lee / Francis T F Tsai /
Abstract: Hsp104 is a ring-forming AAA+ machine that recognizes both aggregated proteins and prion-fibrils as substrates and, together with the Hsp70 system, remodels substrates in an ATP-dependent manner. ...Hsp104 is a ring-forming AAA+ machine that recognizes both aggregated proteins and prion-fibrils as substrates and, together with the Hsp70 system, remodels substrates in an ATP-dependent manner. Whereas the ability to disaggregate proteins is dependent on the Hsp104 M-domain, the location of the M-domain is controversial and its exact function remains unknown. Here we present cryoEM structures of two Hsp104 variants in both crosslinked and noncrosslinked form, in addition to the structure of a functional Hsp104 chimera harboring T4 lysozyme within the M-domain helix L2. Unexpectedly, we found that our Hsp104 chimera has gained function and can solubilize heat-aggregated beta-galactosidase (beta-gal) in the absence of the Hsp70 system. Our fitted structures confirm that the subunit arrangement of Hsp104 is similar to other AAA+ machines, and place the M-domains on the Hsp104 exterior, where they can potentially interact with large, aggregated proteins.
History
DepositionJun 29, 2009-
Header (metadata) releaseMay 7, 2010-
Map releaseMay 7, 2010-
UpdateMay 7, 2010-
Current statusMay 7, 2010Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1631.png

Downloads & links

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Map

FileDownload / File: emd_1631.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHsp104(E285A,E687A) Trap mutant ATP bound state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
1.81 Å/pix.
x 128 pix.
= 231.68 Å		1.81 Å/pix.
x 128 pix.
= 231.68 Å		1.81 Å/pix.
x 128 pix.
= 231.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.81 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-0.259092 - 2.36774
Average (Standard dev.)0.134973 (±0.372228)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 231.68 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.811.811.81
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z231.680231.680231.680
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S213
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.2592.3680.135

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Supplemental data

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Sample components

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Entire : Hsp104

EntireName: Hsp104
Components
  • Sample: Hsp104
  • Protein or peptide: Hsp104

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Supramolecule #1000: Hsp104

SupramoleculeName: Hsp104 / type: sample / ID: 1000 / Oligomeric state: Hexamer / Number unique components: 1
Molecular weightExperimental: 600 KDa / Theoretical: 600 KDa / Method: Calculated from amino acid sequences

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Macromolecule #1: Hsp104

MacromoleculeName: Hsp104 / type: protein_or_peptide / ID: 1 / Name.synonym: Hsp104 / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast / Location in cell: Cytoplasm
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pProEX HTb

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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

Concentration0.4 mg/mL
BufferpH: 7.5 / Details: 50 mM MOPSpH 7.5,2mM DTT, 10mM MgCl2, 5mM ATP
GridDetails: 400 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 83 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot for 5 seconds before plunging

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Electron microscopy

MicroscopeJEOL 2010F
TemperatureAverage: 94 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 400,000 x magnification
Specialist opticsEnergy filter - Name: FEI
DateApr 18, 2008
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Average electron dose: 14 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 60000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 4.7 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: Each CCD image
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: OTHER / Software - Name: EMAN

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