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- PDB-4d2q: Negative-stain electron microscopy of E. coli ClpB mutant E432A (... -

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Basic information

Entry
Database: PDB / ID: 4d2q
TitleNegative-stain electron microscopy of E. coli ClpB mutant E432A (BAP form bound to ClpP)
ComponentsCLPB
KeywordsCHAPERONE / DISAGGREGASE / CLPB / BAP / COILED-COIL DOMAIN
Function / homology
Function and homology information


cellular response to heat / response to heat / protein refolding / ATP hydrolysis activity / ATP binding / membrane / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Chaperonin ClpB / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp amino terminal domain, pathogenicity island component / Clp repeat (R) domain profile. / Clp, repeat (R) domain ...Chaperonin ClpB / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp amino terminal domain, pathogenicity island component / Clp repeat (R) domain profile. / Clp, repeat (R) domain / Clp, N-terminal domain superfamily / ClpA/B family / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chaperone protein ClpB
Similarity search - Component
Biological speciesESCHERICHIA COLI (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 18 Å
AuthorsCarroni, M. / Kummer, E. / Oguchi, Y. / Clare, D.K. / Wendler, P. / Sinning, I. / Kopp, J. / Mogk, A. / Bukau, B. / Saibil, H.R.
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
DepositionMay 12, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 4, 2014Provider: repository / Type: Initial release
Revision 1.1Jun 11, 2014Group: Other
Revision 1.2Aug 23, 2017Group: Data collection / Category: em_software / Item: _em_software.image_processing_id
Remark 650 HELIX DETERMINATION METHOD: AUTHOR PROVIDED.
Remark 700 SHEET DETERMINATION METHOD: AUTHOR PROVIDED.

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-2555
  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: CLPB
B: CLPB
C: CLPB
D: CLPB
E: CLPB
F: CLPB


Theoretical massNumber of molelcules
Total (without water)581,0116
Polymers581,0116
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: Protein
CLPB /


Mass: 96835.086 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Details: THE PROTEIN IS ENGINEERED TO BIND TO CLPP. / Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Plasmid: PDS56 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): MC4100 / References: UniProt: P63284

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: BAP FORM OF CLPB E432A REPRESSED MUTANT WITH ATPGS / Type: COMPLEX
Buffer solutionpH: 7.5
SpecimenConc.: 0.03 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE / Material: uranyl acetate
Specimen supportDetails: CARBON

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Jun 6, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Calibrated magnification: 68000 X / Nominal defocus max: 1200 nm / Nominal defocus min: 500 nm / Cs: 2 mm
Image recordingElectron dose: 20 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
Image scansNum. digital images: 110
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2IMAGIC3D reconstruction
3SPIDER3D reconstruction
CTF correctionDetails: PHASE FLIPPING ENTIRE FRAME
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionMethod: ANGULAR RECONSTITUTION AND PROJECTION MATCHING / Resolution: 18 Å / Num. of particles: 11570
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2555. (DEPOSITION ID: 12235).
Symmetry type: POINT
RefinementHighest resolution: 18 Å
Refinement stepCycle: LAST / Highest resolution: 18 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms14201 0 0 0 14201

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