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- EMDB-2561: Negative-stain electron microscopy of Hsp104 (HAP form bound to ClpP) -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-2561 | |||||||||
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Title | Negative-stain electron microscopy of Hsp104 (HAP form bound to ClpP) | |||||||||
![]() | Reconstruction of Hsp104 (HAP form bound to ClpP). Six fold symmetry applied. | |||||||||
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![]() | chaperone / disaggregase / Hsp104 / HAP / wild type / coiled-coil domain | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 21.0 Å | |||||||||
![]() | Carroni M / Kummer E / Oguchi Y / Clare DK / Wendler P / Sinning I / Kopp J / Mogk A / Bukau B / Saibil HR | |||||||||
![]() | ![]() 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 |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 32.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 10.7 KB 10.7 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 10.6 KB | Display | ![]() |
Images | ![]() | 384 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 2555C ![]() 2556C ![]() 2557C ![]() 2558C ![]() 2559C ![]() 2560C ![]() 2562C ![]() 2563C ![]() 4ciuC ![]() 4d2qC ![]() 4d2uC ![]() 4d2xC C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of Hsp104 (HAP form bound to ClpP). Six fold symmetry applied. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
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Sample components
-Entire : Hsp104 ATPgammaS. HAP variant bound to ClpP.
Entire | Name: Hsp104 ATPgammaS. HAP variant bound to ClpP. |
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Components |
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-Supramolecule #1000: Hsp104 ATPgammaS. HAP variant bound to ClpP.
Supramolecule | Name: Hsp104 ATPgammaS. HAP variant bound to ClpP. / type: sample / ID: 1000 Details: Only the Hsp104 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: Hsp104
Macromolecule | Name: Hsp104 / 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: ![]() ![]() |
Molecular weight | Experimental: 80 KDa / Theoretical: 80 KDa |
Recombinant expression | Organism: ![]() ![]() |
-Experimental details
-Structure determination
Method | negative staining |
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![]() | single particle reconstruction |
Aggregation state | particle |
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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 |
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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 | Oct 30, 2012 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 112 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
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 |
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Image processing
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: ![]() |
Details | An homology model of Hsp104 was created based on the indicated pdb using Phyre2 |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |