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- PDB-6qs8: ClpB (DWB and K476C mutant) bound to casein in presence of ATPgam... -

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Basic information

Entry
Database: PDB / ID: 6qs8
TitleClpB (DWB and K476C mutant) bound to casein in presence of ATPgammaS - state KC-2B
Components
  • Chaperone protein ClpB
  • casein
KeywordsCHAPERONE / disaggregase / proteostasis / AAA
Function / homology
Function and homology information


cellular response to heat / response to heat / protein refolding / ATP hydrolysis activity / ATP binding / membrane / identical protein binding / 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 amino terminal domain, pathogenicity island component / Clp repeat (R) domain profile. / 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 amino terminal domain, pathogenicity island component / Clp repeat (R) domain profile. / Clp, repeat (R) domain / 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
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Chaperone protein ClpB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsDeville, C. / Saibil, H.R.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust106252 United Kingdom
Wellcome Trust101488 United Kingdom
Wellcome Trust079605 United Kingdom
CitationJournal: Cell Rep / Year: 2019
Title: Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor.
Authors: Célia Deville / Kamila Franke / Axel Mogk / Bernd Bukau / Helen R Saibil /
Abstract: AAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and ...AAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and threading activities are regulated and intertwined is key to understanding the AAA+ protein mechanism. We studied the disaggregase ClpB, which contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities. Coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring. Here, we determine the mechanism of ClpB activation by comparing ATPase mechanisms and cryo-EM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant. We show that ClpB activation reduces ATPase cooperativity and induces a sequential mode of ATP hydrolysis in the AAA2 ring, the main ATPase motor. AAA1 and AAA2 rings do not work synchronously but in alternating cycles. This ensures high grip, enabling substrate threading via a processive, rope-climbing mechanism.
History
DepositionFeb 20, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 3, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2019Group: Data collection
Category: em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.2Jul 17, 2019Group: Data collection / Data processing / Category: em_3d_reconstruction / em_software / Item: _em_3d_reconstruction.resolution / _em_software.name
Revision 1.3Aug 21, 2019Group: Data collection / Experimental preparation / Category: em_sample_support / Item: _em_sample_support.grid_type
Revision 1.4Dec 18, 2019Group: 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

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-4627
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Chaperone protein ClpB
B: Chaperone protein ClpB
C: Chaperone protein ClpB
D: Chaperone protein ClpB
E: Chaperone protein ClpB
F: Chaperone protein ClpB
S: casein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)582,16326
Polymers576,3097
Non-polymers5,85419
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area55870 Å2
ΔGint-189 kcal/mol
Surface area145680 Å2
MethodPISA

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Components

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


Mass: 95708.133 Da / Num. of mol.: 6 / Mutation: E279A/K476C/E678A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: clpB, htpM, b2592, JW2573 / Production host: Escherichia coli (E. coli) / References: UniProt: P63284
#2: Protein/peptide casein /


Mass: 2060.531 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle)
#3: 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.: 10 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*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

Component
IDNameTypeEntity IDParent-IDSource
1ClpB-DWB-K476C bound to casein in presence of ATPgammaSCOMPLEX#1-#20MULTIPLE SOURCES
2ClpB-DWB-K476CCOMPLEX#11RECOMBINANT
3caseinCOMPLEX#21NATURAL
Molecular weightValue: 0.57 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Bos taurus (cattle)9913
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HClTrisTris-HClTris1
225 mMpotassium chlorideKCl1
310 mMmagnesium chlorideMgCl21
42 mMadenosine5o3thiotriphosphateATPgammaS1
51 mMdithiothreitolDTT1
SpecimenConc.: 1.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / 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 FIELDBright-field microscopy / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
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) / Num. of grids imaged: 1 / Num. of real images: 6060
EM imaging opticsEnergyfilter name: GIF Bioquantum
Image scansMovie frames/image: 50

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.11.2model fittingrigid body fitting of domains
8Coot0.8.8model fittingbuiling of missing loops and connexions
10PHENIX1.12model refinement
11cryoSPARC1initial Euler assignment
12RELION2.1final Euler assignment
13RELION2.1classification
14RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 875861
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39739 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 1CIU

1ciu

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