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

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Basic information

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


cellular response to heat / response to heat / protein refolding / ATP hydrolysis activity / ATP binding / identical protein binding / membrane / cytosol / cytoplasm
Similarity search - Function
Chaperonin ClpB / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp repeat (R) N-terminal domain / : / Clp, repeat (R) domain ...Chaperonin ClpB / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp repeat (R) N-terminal domain / : / Clp, repeat (R) domain / Clp repeat (R) domain profile. / Clp, N-terminal domain superfamily / ClpA/B family / Clp ATPase, C-terminal / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / AAA domain (Cdc48 subfamily) / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chaperone protein ClpB
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Bos taurus (domestic 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 (NBDs) ...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.
History
DepositionApr 1, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 12, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type
Remark 650HELIX DETERMINATION METHOD: AUTHOR
Remark 700SHEET DETERMINATION METHOD: AUTHOR

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Structure visualization

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  • Deposited structure unit
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  • EMDB-20051
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Structure viewerMolecule:
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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
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein 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 (domestic 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
IDNameTypeEntity IDParent-IDSourceDetails
1hyperactive ClpB mutant K476C bound to caseinCOMPLEXall0MULTIPLE SOURCES
2hyperactive ClpB mutant K476C_NTD trimerCOMPLEX#11RECOMBINANT
3alpha-s1-caseinCOMPLEX#21NATURALProtein 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 (domestic 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 FIELD / 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

1khy


Pdb chain-ID: A / Accession code: 1KHY / Source name: PDB / Type: experimental model

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