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- EMDB-2563: Cryo electron microscopy of E. coli ClpB -

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Basic information

Entry
Database: EMDB / ID: EMD-2563
TitleCryo electron microscopy of E. coli ClpB
Map dataCryo-EM reconstruction of E.coli ClpB. Six fold symmetry applied.
Sample
  • Sample: ClpB with ATPgammaS.
  • Protein or peptide: ClpB
Keywordschaperone / disaggregase / ClpB / coiled-coil domain
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 29.0 Å
AuthorsCarroni M / Kummer E / Oguchi Y / Clare DK / Wendler P / Sinning I / Kopp J / Mogk A / Bukau B / Saibil HR
CitationJournal: Elife / Year: 2014
Title: Head-to-tail interactions of the coiled-coil domains regulate ClpB activity and cooperation with Hsp70 in protein disaggregation.
Authors: Marta Carroni / Eva Kummer / Yuki Oguchi / Petra Wendler / Daniel K Clare / Irmgard Sinning / Jürgen Kopp / Axel Mogk / Bernd Bukau / Helen R Saibil /
Abstract: The hexameric AAA+ chaperone ClpB reactivates aggregated proteins in cooperation with the Hsp70 system. Essential for disaggregation, the ClpB middle domain (MD) is a coiled-coil propeller that binds ...The hexameric AAA+ chaperone ClpB reactivates aggregated proteins in cooperation with the Hsp70 system. Essential for disaggregation, the ClpB middle domain (MD) is a coiled-coil propeller that binds Hsp70. Although the ClpB subunit structure is known, positioning of the MD in the hexamer and its mechanism of action are unclear. We obtained electron microscopy (EM) structures of the BAP variant of ClpB that binds the protease ClpP, clearly revealing MD density on the surface of the ClpB ring. Mutant analysis and asymmetric reconstructions show that MDs adopt diverse positions in a single ClpB hexamer. Adjacent, horizontally oriented MDs form head-to-tail contacts and repress ClpB activity by preventing Hsp70 interaction. Tilting of the MD breaks this contact, allowing Hsp70 binding, and releasing the contact in adjacent subunits. Our data suggest a wavelike activation of ClpB subunits around the ring.DOI: http://dx.doi.org/10.7554/eLife.02481.001.
History
DepositionJan 16, 2014-
Header (metadata) releaseFeb 19, 2014-
Map releaseMay 14, 2014-
UpdateJun 25, 2014-
Current statusJun 25, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.213
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.213
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2563.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of E.coli ClpB. Six fold symmetry applied.
Voxel sizeX=Y=Z: 2 Å
Density
Contour LevelBy AUTHOR: 0.213 / Movie #1: 0.213
Minimum - Maximum-1.70340157 - 5.54236603
Average (Standard dev.)0.00092725 (±0.30020744)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-63-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 256.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z256.000256.000256.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS-64-63-64
NC/NR/NS128128128
D min/max/mean-1.7035.5420.001

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Supplemental data

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Sample components

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Entire : ClpB with ATPgammaS.

EntireName: ClpB with ATPgammaS.
Components
  • Sample: ClpB with ATPgammaS.
  • Protein or peptide: ClpB

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Supramolecule #1000: ClpB with ATPgammaS.

SupramoleculeName: ClpB with ATPgammaS. / type: sample / ID: 1000 / Oligomeric state: Homohexamer. / Number unique components: 1
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: ClpB

MacromoleculeName: ClpB / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: cytoplasm
Molecular weightExperimental: 80 KDa / Theoretical: 80 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: derivatives of MC4100 / Recombinant plasmid: pDS56

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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 Tris-HCl, pH 7.5, 20 mM KCl, 15 mM MgCl2, 1 mM DTT, 2 mM ATPgammaS
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 80000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureMin: 77 K / Max: 88 K / Average: 83 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 x magnification
DateFeb 4, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 200 / Average electron dose: 15 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: phase flipping entire frame
Final angle assignmentDetails: Only side views used selected by using MSA and classification. Beta angles between 80 and 100 degrees (IMAGIC convention).
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 29.0 Å / Resolution method: OTHER / Software - Name: IMAGIC, Spider
Details: Starting models were generated by angular reconstitution and particle orientations were refined by projection matching in SPIDER.
Number images used: 7606
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Chain ID: A
SoftwareName: Chimera
DetailsFitting of separate domains was performed manually and locally optimised using Chimera. Known domain interfaces were used to guide the fit.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

Chain - Chain ID: A
SoftwareName: Chimera
DetailsFitting of separate domains was performed manually and locally optimised using Chimera. Known domain interfaces were used to guide the fit.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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