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Yorodumi- EMDB-1359: Atypical AAA+ subunit packing creates an expanded cavity for disa... -
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-Basic information
Entry | Database: EMDB / ID: EMD-1359 | |||||||||
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Title | Atypical AAA+ subunit packing creates an expanded cavity for disaggregation by the protein-remodeling factor Hsp104. | |||||||||
Map data | Hsp104 deltaN 3D density map. Hexamer bound to ATPgammaS. | |||||||||
Sample |
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Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 11.0 Å | |||||||||
Authors | Wendler P / Shorter J / Plisson C / Cashikar AG / Lindquist S / Saibil HR | |||||||||
Citation | Journal: Cell / Year: 2007 Title: Atypical AAA+ subunit packing creates an expanded cavity for disaggregation by the protein-remodeling factor Hsp104. Authors: Petra Wendler / James Shorter / Celia Plisson / Anil G Cashikar / Susan Lindquist / Helen R Saibil / Abstract: Hsp104, a yeast protein-remodeling factor of the AAA+ (ATPases associated with various cellular activities) superfamily, and its homologs in bacteria and plants mediate cell recovery after severe ...Hsp104, a yeast protein-remodeling factor of the AAA+ (ATPases associated with various cellular activities) superfamily, and its homologs in bacteria and plants mediate cell recovery after severe stress by disaggregating denatured proteins through a poorly understood mechanism. Here, we present cryo-electron microscopy maps and domain fitting of Hsp104 hexamers, revealing an unusual arrangement of AAA+ modules with the prominent coiled-coil domain intercalated between the AAA+ domains. This packing results in a greatly expanded cavity, which is capped at either end by N- and C-terminal domains. The fitted structures as well as mutation of conserved coiled-coil arginines suggest that the coiled-coil domain plays a major role in the extraction of proteins from aggregates, providing conserved residues for key functions in ATP hydrolysis and potentially for substrate interaction. The large cavity could enable the uptake of polypeptide loops without a requirement for exposed N or C termini. | |||||||||
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_1359.map.gz | 459.2 KB | EMDB map data format | |
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Header (meta data) | emd-1359-v30.xml emd-1359.xml | 10 KB 10 KB | Display Display | EMDB header |
Images | 1359.gif | 184.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1359 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1359 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_1359.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Hsp104 deltaN 3D density map. Hexamer bound to ATPgammaS. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.8 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 : Hsp104 deltaN ATPgammaS
Entire | Name: Hsp104 deltaN ATPgammaS |
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Components |
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-Supramolecule #1000: Hsp104 deltaN ATPgammaS
Supramolecule | Name: Hsp104 deltaN ATPgammaS / type: sample / ID: 1000 / Oligomeric state: Hexamer / Number unique components: 2 |
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Molecular weight | Experimental: 500 KDa / Theoretical: 500 KDa |
-Supramolecule #1: Hsp104 deltaN
Supramolecule | Name: Hsp104 deltaN / type: organelle_or_cellular_component / ID: 1 / Name.synonym: Heat Shock Protein 104 Details: The Hsp104 fragment comprises aa residues 157 to 908. Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast |
Molecular weight | Experimental: 83 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pPROEX-HTb-Hsp104 |
-Macromolecule #1: ATPgammaS
Macromolecule | Name: ATPgammaS / type: em_label / ID: 1 |
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-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.3 mg/mL |
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Buffer | pH: 7.5 Details: 20 mM HEPES pH 7.5, 20 mM NaCl, 10 mM MgCl2, 1mM DTT, 2mM ATPgammaS |
Grid | Details: 300 mesh copper grid- holey carbon film |
Vitrification | Cryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: home made Method: The grids were blotted for 2-3 seconds and then immediately plunged into liquid ethane |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.1 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: single tilt cryo / Specimen holder model: GATAN LIQUID NITROGEN |
Temperature | Min: 77 K / Max: 85 K / Average: 77 K |
Alignment procedure | Legacy - Astigmatism: objective lens astigmatism was corrected for at 150,000 x magnification |
Date | Jan 18, 2005 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 19 / Average electron dose: 15 e/Å2 / Od range: 1 / Bits/pixel: 8 |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: phase flipping, each particle |
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Final reconstruction | Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Imagic, Spider / Number images used: 3623 |
-Atomic model buiding 1
Details | The domains were fitted manually as rigid bodies using Pymol. Automated fitting in Chimera optimised the fit for NBD2. |
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Refinement | Protocol: RIGID BODY FIT |