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- PDB-5ofo: Cryo EM structure of the E. coli disaggregase ClpB (BAP form, DWB... -

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Basic information

Entry
Database: PDB / ID: 5ofo
TitleCryo EM structure of the E. coli disaggregase ClpB (BAP form, DWB mutant), in the ATPgammaS state, bound to the model substrate casein
ComponentsChaperone protein ClpB,ATP-dependent Clp protease ATP-binding subunit ClpA,Chaperone protein ClpB
KeywordsCHAPERONE / disaggregase / ClpB / AAA
Function / homology
Function and homology information


endopeptidase Clp complex / protein quality control for misfolded or incompletely synthesized proteins / ATP-dependent peptidase activity / protein unfolding / cellular response to heat / response to heat / protein refolding / response to oxidative stress / ATP hydrolysis activity / proteolysis ...endopeptidase Clp complex / protein quality control for misfolded or incompletely synthesized proteins / ATP-dependent peptidase activity / protein unfolding / cellular response to heat / response to heat / protein refolding / response to oxidative stress / ATP hydrolysis activity / proteolysis / ATP binding / identical protein binding / membrane / cytosol / cytoplasm
Similarity search - Function
ATP-dependent Clp protease ATP-binding subunit ClpA / 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 amino terminal domain, pathogenicity island component / : ...ATP-dependent Clp protease ATP-binding subunit ClpA / 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 amino terminal domain, pathogenicity island component / : / Clp, repeat (R) domain / Clp repeat (R) domain profile. / Clp, N-terminal domain superfamily / ClpA/B family / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / 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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-dependent Clp protease ATP-binding subunit ClpA / Chaperone protein ClpB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsDeville, C. / Carroni, M. / Franke, K.B. / Topf, M. / Bukau, B. / Mogk, A. / Saibil, H.R.
CitationJournal: Sci Adv / Year: 2017
Title: Structural pathway of regulated substrate transfer and threading through an Hsp100 disaggregase.
Authors: Célia Deville / Marta Carroni / Kamila B Franke / Maya Topf / Bernd Bukau / Axel Mogk / Helen R Saibil /
Abstract: Refolding aggregated proteins is essential in combating cellular proteotoxic stress. Together with Hsp70, Hsp100 chaperones, including ClpB, form a powerful disaggregation machine that threads ...Refolding aggregated proteins is essential in combating cellular proteotoxic stress. Together with Hsp70, Hsp100 chaperones, including ClpB, form a powerful disaggregation machine that threads aggregated polypeptides through the central pore of tandem adenosine triphosphatase (ATPase) rings. To visualize protein disaggregation, we determined cryo-electron microscopy structures of inactive and substrate-bound ClpB in the presence of adenosine 5'--(3-thiotriphosphate), revealing closed AAA+ rings with a pronounced seam. In the substrate-free state, a marked gradient of resolution, likely corresponding to mobility, spans across the AAA+ rings with a dynamic hotspot at the seam. On the seam side, the coiled-coil regulatory domains are locked in a horizontal, inactive orientation. On the opposite side, the regulatory domains are accessible for Hsp70 binding, substrate targeting, and activation. In the presence of the model substrate casein, the polypeptide threads through the entire pore channel and increased nucleotide occupancy correlates with higher ATPase activity. Substrate-induced domain displacements indicate a pathway of regulated substrate transfer from Hsp70 to the ClpB pore, inside which a spiral of loops contacts the substrate. The seam pore loops undergo marked displacements, along with ordering of the regulatory domains. These asymmetric movements suggest a mechanism for ATPase activation and substrate threading during disaggregation.
History
DepositionJul 11, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 16, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2017Group: Database references / Category: citation
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Aug 21, 2019Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.3Oct 23, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.4Nov 6, 2019Group: Data collection / Database references / Category: pdbx_database_related / Item: _pdbx_database_related.content_type
Revision 1.5May 8, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
C: Chaperone protein ClpB,ATP-dependent Clp protease ATP-binding subunit ClpA,Chaperone protein ClpB
F: Chaperone protein ClpB,ATP-dependent Clp protease ATP-binding subunit ClpA,Chaperone protein ClpB
E: Chaperone protein ClpB,ATP-dependent Clp protease ATP-binding subunit ClpA,Chaperone protein ClpB
D: Chaperone protein ClpB,ATP-dependent Clp protease ATP-binding subunit ClpA,Chaperone protein ClpB
B: Chaperone protein ClpB,ATP-dependent Clp protease ATP-binding subunit ClpA,Chaperone protein ClpB
A: Chaperone protein ClpB,ATP-dependent Clp protease ATP-binding subunit ClpA,Chaperone protein ClpB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)589,48917
Polymers583,7336
Non-polymers5,75611
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Complex stable by gel filtration and stable on EM grids
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area47030 Å2
ΔGint-48 kcal/mol
Surface area192730 Å2
MethodPISA

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Components

#1: Protein
Chaperone protein ClpB,ATP-dependent Clp protease ATP-binding subunit ClpA,Chaperone protein ClpB / Heat shock protein F84.1


Mass: 97288.914 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria), (gene. exp.) Escherichia coli (E. coli)
Strain: K12 / Gene: clpB, htpM, b2592, JW2573, clpA, lopD, b0882, JW0866 / Production host: Escherichia coli (E. coli) / References: UniProt: P63284, UniProt: P0ABH9
#2: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM

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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

ComponentName: ClpB, BAP form, double walker B mutant / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.57 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
125 mmol/LtrisTrisHCl1
225 mmol/Lsodium chlorideNaCl1
310 mmol/Lmagnesium chlorideMgCl21
42 mmol/Ladenosine 5'-O-(3-thio)triphosphateATPgammaS1
51 mmol/LdithiothreitolDTT1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Agar lacey carbon
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Chamber temperature: 295 K

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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 / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansSampling size: 5 µm / Movie frames/image: 50

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1Gautomatchparticle selection
2EPUimage acquisition
4CTFFIND4CTF correctionfor CTF estimation
7iMODFITmodel fitting
8Flex-EMmodel fitting
10RELION2initial Euler assignment
11RELION2final Euler assignment
12RELION2classification
13RELION23D reconstruction
14PHENIX1.11.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 500000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 230000 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Local correlation coefficient
Details: Initial models of the monomer were generated using MODELLER v9.17 with previously determined crystal structures of ClpB or ClpB domains as templates (PDB ids 4CIU, 1QVR, 4HSE and 4LJ9). The ...Details: Initial models of the monomer were generated using MODELLER v9.17 with previously determined crystal structures of ClpB or ClpB domains as templates (PDB ids 4CIU, 1QVR, 4HSE and 4LJ9). The crystal structure of E. coli ClpB (4CIU) was used as a main template, the crystal structure of T. thermophilus (1QVR) was used to model positions of the NTD and the crystal structures of AAA1 and AAA2 of E. coli ClpB (4HSE and 4LJ9) were used to model the pore loops disordered in other crystal structures. Initial rigid body fitting of the monomers in the map was manually done in Chimera using the Fit-in-Map tool. iMODFIT was used for fitting involving large domain motions. FlexEM was then used for refinement of secondary structures and loops in the map. Quality and improvement of the fit was assessed with TEMPy using the Segment based Manders Overlap Coefficient (SMOC) scores and segment based cross correlation scores. Ridig body fitting of ATPgammaS (structure extracted from PDB id 3EIH) into the nucleotide biding sides was manually done in Chimera using the Fit-in-Map tool. The target map for fitting of ATPgammaS was the difference map between experimental map and map generated using the nucleotide-free protein model revealing nucleotide densities. A round of real-space refinement was performed in phenix using energy minimization to fix the model's geometry and clashes.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00833963
ELECTRON MICROSCOPYf_angle_d1.11245806
ELECTRON MICROSCOPYf_dihedral_angle_d6.98521099
ELECTRON MICROSCOPYf_chiral_restr0.0585160
ELECTRON MICROSCOPYf_plane_restr0.0085999

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