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Yorodumi- EMDB-2559: Negative-stain electron microscopy of E. coli ClpB (BAP form boun... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2559 | |||||||||
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Title | Negative-stain electron microscopy of E. coli ClpB (BAP form bound to ClpP) | |||||||||
Map data | Reconstruction of the E.coli ClpB hyperactive mutant Y503D (BAP form bound to ClpP). Six fold symmetry applied. | |||||||||
Sample |
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Keywords | chaperone / disaggregase / ClpB / BAP / Y503D hyperactive mutant / coiled-coil domain | |||||||||
Function / homology | Function and homology information cellular response to heat / response to heat / protein refolding / ATP hydrolysis activity / ATP binding / identical protein binding / membrane / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 20.0 Å | |||||||||
Authors | Carroni M / Kummer E / Oguchi Y / Clare DK / Wendler P / Sinning I / Kopp J / Mogk A / Bukau B / Saibil HR | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2559.map.gz | 32.1 MB | EMDB map data format | |
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Header (meta data) | emd-2559-v30.xml emd-2559.xml | 11.8 KB 11.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_2559_fsc.xml | 10.6 KB | Display | FSC data file |
Images | emd_2559.tiff | 415.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2559 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2559 | HTTPS FTP |
-Validation report
Summary document | emd_2559_validation.pdf.gz | 220.5 KB | Display | EMDB validaton report |
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Full document | emd_2559_full_validation.pdf.gz | 219.6 KB | Display | |
Data in XML | emd_2559_validation.xml.gz | 10.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2559 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2559 | HTTPS FTP |
-Related structure data
Related structure data | 4d2xMC 2555C 2556C 2557C 2558C 2560C 2561C 2562C 2563C 4ciuC 4d2qC 4d2uC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_2559.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of the E.coli ClpB hyperactive mutant Y503D (BAP form bound to ClpP). Six fold symmetry applied. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : ClpB Y503D mutant with ATPgammaS. BAP variant bound to ClpP.
Entire | Name: ClpB Y503D mutant with ATPgammaS. BAP variant bound to ClpP. |
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Components |
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-Supramolecule #1000: ClpB Y503D mutant with ATPgammaS. BAP variant bound to ClpP.
Supramolecule | Name: ClpB Y503D mutant with 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 |
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Molecular weight | Theoretical: 500 KDa |
-Macromolecule #1: ClpB
Macromolecule | Name: 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 |
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Source (natural) | Organism: Escherichia coli (E. coli) / Location in cell: cytoplasm |
Molecular weight | Experimental: 80 KDa / Theoretical: 80 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: derivatives of MC4100 / Recombinant plasmid: pDS56 |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.03 mg/mL |
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Buffer | pH: 7.5 Details: 20 mM Tris-HCl, pH 7.5, 20 mM KCl, 15 mM MgCl2, 1 mM DTT, 2 mM ATPgammaS |
Staining | Type: NEGATIVE Details: protein adsorbed on carbon coated grids pretreated with 0.01% poly lysine chains. Stained with 2% uranyl acetate for 1 minute. |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 x magnification |
Date | Jul 20, 2011 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 108 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated 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 stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: Chimera |
Details | Fitting of separate domains was performed manually and locally optimised using Chimera. Known domain interfaces were used to guide the fit. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | PDB-4d2x: |
-Atomic model buiding 2
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: Chimera |
Details | Fitting of separate domains was performed manually and locally optimised using Chimera. Known domain interfaces were used to guide the fit. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | PDB-4d2x: |