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- EMDB-2556: Negative-stain electron microscopy of E. coli ClpB mutant E432A (... -

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Basic information

Entry
Database: EMDB / ID: EMD-2556
TitleNegative-stain electron microscopy of E. coli ClpB mutant E432A (BAP form bound to ClpP)
Map dataAsymmetric reconstruction of ClpB E432A mutant (BAP form bound to CLpP)
Sample
  • Sample: ClpB E432A ATPgammaS. BAP variant bound to ClpP.
  • Protein or peptide: ClpB
Keywordschaperone / disaggregase / ClpB / BAP / repressed E432A mutant / coiled-coil domain
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 Å
AuthorsCarroni M / Kummer E / Oguchi Y / 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: 0.314
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.314
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2556.tiff

Downloads & links

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Map

FileDownload / File: emd_2556.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAsymmetric reconstruction of ClpB E432A mutant (BAP form bound to CLpP)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 256 pix.
= 512. Å		2 Å/pix.
x 256 pix.
= 512. Å		2 Å/pix.
x 256 pix.
= 512. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.314 / Movie #1: 0.314
Minimum - Maximum-1.19673824 - 8.99914455
Average (Standard dev.)0.00858378 (±0.15903173)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-127-127-127
Dimensions256256256
Spacing256256256
CellA=B=C: 512.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z512.000512.000512.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS-127-127-127
NC/NR/NS256256256
D min/max/mean-1.1978.9990.009

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Supplemental data

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Sample components

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Entire : ClpB E432A ATPgammaS. BAP variant bound to ClpP.

EntireName: ClpB E432A ATPgammaS. BAP variant bound to ClpP.
Components
  • Sample: ClpB E432A ATPgammaS. BAP variant bound to ClpP.
  • Protein or peptide: ClpB

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Supramolecule #1000: ClpB E432A ATPgammaS. BAP variant bound to ClpP.

SupramoleculeName: ClpB E432A ATPgammaS. BAP variant bound to ClpP. / type: sample / ID: 1000
Details: Only the ClpB part was reconstructed and the molecular weight only refers to this part.
Oligomeric state: Homohexamer. (One homohexamer of BAP bound to one homoheptamer of ClpP)
Number unique components: 1
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: ClpB

MacromoleculeName: ClpB / type: protein_or_peptide / ID: 1 / Details: The protein is engineered to bind to ClpP. / 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

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.03 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
StainingType: NEGATIVE
Details: protein adsorbed on carbon coated grids pretreated with 0.01% poly lysine chains. Stained with 2% uranyl acetate for 1 minute.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 x magnification
DateJun 6, 2011
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 110 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 68000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: phase flipping entire frame
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 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. Only part of the molecule was refined in the alignment, but ...Details: Starting models were generated by angular reconstitution and particle orientations were refined by projection matching in SPIDER. Only part of the molecule was refined in the alignment, but the final reconstruction includes the whole molecule.
Number images used: 9288
Final angle assignmentDetails: Only side views used. Beta angles between 80 and 100 degrees (IMAGIC convention).
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1khy


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:

4ciu


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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