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- PDB-6og3: Focus classification structure of the hyperactive ClpB mutant K47... -

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Entry
Database: PDB / ID: 6og3
TitleFocus classification structure of the hyperactive ClpB mutant K476C, bound to casein, NTD-trimer
Components
  • Alpha S1-casein
  • Hyperactive disaggregase ClpB
KeywordsCHAPERONE / disaggregase / CLPB / AAA+
Function / homology
Function and homology information


protein metabolic process / response to unfolded protein / ATPase activity, coupled / response to heat / protein refolding / membrane / ATP binding / identical protein binding / cytosol
ClpA/B, conserved site 1 / Chaperonin ClpB / AAA domain (Cdc48 subfamily) / Clp amino terminal domain, pathogenicity island component / ATPase family associated with various cellular activities (AAA) / ClpA/ClpB, AAA lid domain / Clp, N-terminal domain superfamily / ClpA/B, conserved site 2 / P-loop containing nucleoside triphosphate hydrolase / Clp ATPase, C-terminal ...ClpA/B, conserved site 1 / Chaperonin ClpB / AAA domain (Cdc48 subfamily) / Clp amino terminal domain, pathogenicity island component / ATPase family associated with various cellular activities (AAA) / ClpA/ClpB, AAA lid domain / Clp, N-terminal domain superfamily / ClpA/B, conserved site 2 / P-loop containing nucleoside triphosphate hydrolase / Clp ATPase, C-terminal / Chaperonins clpA/B signature 1. / Clp, N-terminal / AAA lid domain / ATPase, AAA-type, core / AAA+ ATPase domain / ClpA/B family / Chaperonins clpA/B signature 2. / C-terminal, D2-small domain, of ClpB protein
Chaperone protein ClpB
Biological speciesEscherichia coli K-12 (bacteria)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsRizo, A.R. / Lin, J.-B. / Gates, S.N. / Tse, E. / Bart, S.M. / Castellano, L.M. / Dimaio, F. / Shorter, J. / Southworth, D.R.
CitationJournal: 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 1, 2019 / Release: Jun 12, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jun 12, 2019Structure modelrepositoryInitial release
1.1Jun 19, 2019Structure modelData collection / Database referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Remark 650HELIX DETERMINATION METHOD: AUTHOR
Remark 700SHEET DETERMINATION METHOD: AUTHOR

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Assembly

Deposited unit
E: Hyperactive disaggregase ClpB
C: Hyperactive disaggregase ClpB
A: Hyperactive disaggregase ClpB
P: Alpha S1-casein


Theoretical massNumber of molelcules
Total (without water)291,6654
Polymers291,6654
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide Hyperactive disaggregase ClpB / Chaperone protein ClpB / Heat shock protein F84.1


Mass: 96960.367 Da / Num. of mol.: 3 / Mutation: K476C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: clpB, htpM, b2592, JW2573 / Production host: Escherichia coli (E. coli) / References: UniProt: P63284
#2: Protein/peptide Alpha S1-casein


Mass: 783.958 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle)

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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

Component

Type: COMPLEX

IDNameEntity IDParent-IDSourceDetails
1hyperactive ClpB mutant K476C bound to casein1, 20MULTIPLE SOURCES
2hyperactive ClpB mutant K476C_NTD trimer11RECOMBINANT
3alpha-s1-casein21NATURALProtein was purchased from Sigma-Aldrich. Purified from bovine milk.
Molecular weightExperimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23Bos taurus (cattle)9913
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.6 mm
Image recordingElectron dose: 56 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93588 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 1KHY
Pdb chain-ID: A

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