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- EMDB-1244: Visualizing the ATPase cycle in a protein disaggregating machine:... -

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Basic information

Entry
Database: EMDB / ID: EMD-1244
TitleVisualizing the ATPase cycle in a protein disaggregating machine: structural basis for substrate binding by ClpB.
Map dataVolume file of TClpB trap mutant in ATP bound state
Sample
  • Sample: ClpB Trap mutant with E271A and E668A double mutation
  • Protein or peptide: ClpB
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.2 Å
AuthorsTsai F
CitationJournal: Mol Cell / Year: 2007
Title: Visualizing the ATPase cycle in a protein disaggregating machine: structural basis for substrate binding by ClpB.
Authors: Sukyeong Lee / Jae-Mun Choi / Francis T F Tsai /
Abstract: ClpB is a ring-shaped molecular chaperone that has the remarkable ability to disaggregate stress-damaged proteins. Here we present the electron cryomicroscopy reconstruction of an ATP-activated ClpB ...ClpB is a ring-shaped molecular chaperone that has the remarkable ability to disaggregate stress-damaged proteins. Here we present the electron cryomicroscopy reconstruction of an ATP-activated ClpB trap mutant, along with reconstructions of ClpB in the AMPPNP, ADP, and in the nucleotide-free state. We show that motif 2 of the ClpB M domain is positioned between the D1-large domains of neighboring subunits and could facilitate a concerted, ATP-driven conformational change in the AAA-1 ring. We further demonstrate biochemically that ATP is essential for high-affinity substrate binding to ClpB and cannot be substituted with AMPPNP. Our structures show that in the ATP-activated state, the D1 loops are stabilized at the central pore, providing the structural basis for high-affinity substrate binding. Taken together, our results support a mechanism by which ClpB captures substrates on the upper surface of the AAA-1 ring before threading them through the ClpB hexamer in an ATP hydrolysis-driven step.
History
DepositionJul 13, 2006-
Header (metadata) releaseJul 13, 2006-
Map releaseMar 27, 2007-
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: 0.93246
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.93246
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1244.gif

Downloads & links

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Map

FileDownload / File: emd_1244.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVolume file of TClpB trap mutant in ATP bound state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.17 Å/pix.
x 96 pix.
= 208.608 Å		2.17 Å/pix.
x 96 pix.
= 208.608 Å		2.17 Å/pix.
x 96 pix.
= 208.608 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.173 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 1.62 / Movie #1: 0.93246
Minimum - Maximum-0.796974 - 5.93084
Average (Standard dev.)0.193313 (±0.627797)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-48-48-48
Dimensions969696
Spacing969696
CellA=B=C: 208.608 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.1732.1732.173
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z208.608208.608208.608
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-48-48-48
NC/NR/NS969696
D min/max/mean-0.7975.9310.193

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Supplemental data

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Sample components

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Entire : ClpB Trap mutant with E271A and E668A double mutation

EntireName: ClpB Trap mutant with E271A and E668A double mutation
Components
  • Sample: ClpB Trap mutant with E271A and E668A double mutation
  • Protein or peptide: ClpB

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Supramolecule #1000: ClpB Trap mutant with E271A and E668A double mutation

SupramoleculeName: ClpB Trap mutant with E271A and E668A double mutation / type: sample / ID: 1000 / Oligomeric state: homohexamer / Number unique components: 1
Molecular weightExperimental: 600 KDa / Theoretical: 600 KDa

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Macromolecule #1: ClpB

MacromoleculeName: ClpB / type: protein_or_peptide / ID: 1
Details: Trap mutant was generated by introducing double Walker B mutations: E271A and E668A. Trap mutant can bind ATP but not able to hydrolize ATP.
Number of copies: 6 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Thermus thermophilus (bacteria) / Cell: E.coli / Location in cell: cytoplasm
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET24a

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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

Concentration0.03 mg/mL
BufferpH: 7.5 / Details: 50mM MOPS,150mM KCl, 5mM MgCl2, 2mM ATP
GridDetails: 400 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber temperature: 90 K / Instrument: REICHERT-JUNG PLUNGER / Details: Vitrification instrument: Reichert plunger

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Electron microscopy

MicroscopeJEOL 2010F
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 400,000x magnification
DateJul 6, 2004
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 24 / Average electron dose: 15 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.0 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 6900
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: each CCD frame
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 19008

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