|Entry||Database: PDB / ID: 6og2|
|Title||Focus classification structure of the hyperactive ClpB mutant K476C, bound to casein, post-state|
|Components||Hyperactive disaggregase ClpB|
|Keywords||CHAPERONE / disaggregase / CLPB / AAA+|
|Biological species||Escherichia coli K-12 (bacteria)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å|
|Authors||Rizo, A.R. / Lin, J.-B. / Gates, S.N. / Tse, E. / Bart, S.M. / Castellano, L.M. / Dimaio, F. / Shorter, J. / Southworth, D.R.|
|Citation||Journal: Nat Commun / Year: 2019|
Title: Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase.
Authors: Alexandrea N Rizo / JiaBei Lin / Stephanie N Gates / Eric Tse / Stephen M Bart / Laura M Castellano / Frank DiMaio / James Shorter / Daniel R Southworth /
Abstract: Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains ...Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. Here we report cryo-EM structures of a hyperactive ClpB variant bound to the model substrate, casein in the presence of slowly hydrolysable ATPγS, which reveal the translocation mechanism. Distinct substrate-gripping interactions are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent to the topmost NBD1 contact. NBD conformations at the seam interface reveal how ATP hydrolysis-driven substrate disengagement and re-binding are precisely tuned to drive a directional, stepwise translocation cycle.
SummaryFull reportAbout validation report
|Date||Deposition: Apr 1, 2019 / Release: Jun 12, 2019|
|Remark 650||HELIX DETERMINATION METHOD: AUTHOR|
|Remark 700||SHEET DETERMINATION METHOD: AUTHOR|
|Structure viewer||Molecule: |
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F: Hyperactive disaggregase ClpB
A: Hyperactive disaggregase ClpB
Mass: 97018.469 Da / Num. of mol.: 2 / Mutation: K476C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Production host: Escherichia coli (E. coli)
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: hyperactive disaggregase ClpB K476C, bound to casein / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT|
|Molecular weight||Experimental value: YES|
|Source (natural)||Organism: Escherichia coli K-12 (bacteria)|
|Source (recombinant)||Organism: Escherichia coli (E. coli)|
|Buffer solution||pH: 7.4|
|Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Details: unspecified|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Image recording||Electron dose: 56 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Symmetry||Point symmetry: C1 (asymmetric)|
|3D reconstruction||Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91601 / Symmetry type: POINT|
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