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- PDB-6w20: ClpAP Disengaged State bound to RepA-GFP -

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Basic information

Entry
Database: PDB / ID: 6w20
TitleClpAP Disengaged State bound to RepA-GFP
Components
  • ATP-dependent Clp protease ATP-binding subunit ClpA
  • ATP-dependent Clp protease proteolytic subunit
  • RepA, green fluorescent protein fusion
KeywordsCHAPERONE / AAA+ / Protease / Hsp100 / ATPase
Function / homology
Function and homology information


HslUV protease complex / endopeptidase Clp / endopeptidase Clp complex / positive regulation of programmed cell death / response to temperature stimulus / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / protein unfolding / proteasomal protein catabolic process / serine-type peptidase activity ...HslUV protease complex / endopeptidase Clp / endopeptidase Clp complex / positive regulation of programmed cell death / response to temperature stimulus / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / protein unfolding / proteasomal protein catabolic process / serine-type peptidase activity / response to radiation / cellular response to heat / ATPase binding / response to heat / response to oxidative stress / serine-type endopeptidase activity / ATP hydrolysis activity / proteolysis / ATP binding / identical protein binding / membrane / cytoplasm / cytosol
Similarity search - Function
ATP-dependent Clp protease ATP-binding subunit ClpA / ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpP, histidine active site / Endopeptidase Clp histidine active site. / ATP-dependent Clp protease proteolytic subunit / Clp protease proteolytic subunit /Translocation-enhancing protein TepA / Clp protease ...ATP-dependent Clp protease ATP-binding subunit ClpA / ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpP, histidine active site / Endopeptidase Clp histidine active site. / ATP-dependent Clp protease proteolytic subunit / Clp protease proteolytic subunit /Translocation-enhancing protein TepA / Clp protease / 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 / 2-enoyl-CoA Hydratase; Chain A, domain 1 / 2-enoyl-CoA Hydratase; Chain A, domain 1 / ClpP/crotonase-like domain superfamily / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Alpha-Beta Complex / P-loop containing nucleoside triphosphate hydrolase / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease ATP-binding subunit ClpA / ATP-dependent Clp protease proteolytic subunit
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsLopez, K.L. / Rizo, A.N. / Tse, E. / Lin, J. / Scull, N.W. / Thwin, A.C. / Lucius, A.L. / Shorter, J. / Southworth, D.R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Conformational plasticity of the ClpAP AAA+ protease couples protein unfolding and proteolysis.
Authors: Kyle E Lopez / Alexandrea N Rizo / Eric Tse / JiaBei Lin / Nathaniel W Scull / Aye C Thwin / Aaron L Lucius / James Shorter / Daniel R Southworth /
Abstract: The ClpAP complex is a conserved bacterial protease that unfolds and degrades proteins targeted for destruction. The ClpA double-ring hexamer powers substrate unfolding and translocation into the ...The ClpAP complex is a conserved bacterial protease that unfolds and degrades proteins targeted for destruction. The ClpA double-ring hexamer powers substrate unfolding and translocation into the ClpP proteolytic chamber. Here, we determined high-resolution structures of wild-type Escherichia coli ClpAP undergoing active substrate unfolding and proteolysis. A spiral of pore loop-substrate contacts spans both ClpA AAA+ domains. Protomers at the spiral seam undergo nucleotide-specific rearrangements, supporting substrate translocation. IGL loops extend flexibly to bind the planar, heptameric ClpP surface with the empty, symmetry-mismatched IGL pocket maintained at the seam. Three different structures identify a binding-pocket switch by the IGL loop of the lowest positioned protomer, involving release and re-engagement with the clockwise pocket. This switch is coupled to a ClpA rotation and a network of conformational changes across the seam, suggesting that ClpA can rotate around the ClpP apical surface during processive steps of translocation and proteolysis.
History
DepositionMar 4, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 6, 2020Provider: repository / Type: Initial release
Revision 1.1May 13, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2May 27, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Mar 6, 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
G: ATP-dependent Clp protease proteolytic subunit
H: ATP-dependent Clp protease proteolytic subunit
I: ATP-dependent Clp protease proteolytic subunit
J: ATP-dependent Clp protease proteolytic subunit
K: ATP-dependent Clp protease proteolytic subunit
L: ATP-dependent Clp protease proteolytic subunit
M: ATP-dependent Clp protease proteolytic subunit
N: ATP-dependent Clp protease proteolytic subunit
O: ATP-dependent Clp protease proteolytic subunit
P: ATP-dependent Clp protease proteolytic subunit
Q: ATP-dependent Clp protease proteolytic subunit
R: ATP-dependent Clp protease proteolytic subunit
S: ATP-dependent Clp protease proteolytic subunit
T: ATP-dependent Clp protease proteolytic subunit
A: ATP-dependent Clp protease ATP-binding subunit ClpA
B: ATP-dependent Clp protease ATP-binding subunit ClpA
C: ATP-dependent Clp protease ATP-binding subunit ClpA
D: ATP-dependent Clp protease ATP-binding subunit ClpA
E: ATP-dependent Clp protease ATP-binding subunit ClpA
F: ATP-dependent Clp protease ATP-binding subunit ClpA
X: RepA, green fluorescent protein fusion
hetero molecules


Theoretical massNumber of molelcules
Total (without water)838,30832
Polymers832,96921
Non-polymers5,33911
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area120280 Å2
ΔGint-370 kcal/mol
Surface area221010 Å2

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Components

#1: Protein
ATP-dependent Clp protease proteolytic subunit / Endopeptidase Clp


Mass: 23212.650 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: clpP, A1WS_00920 / Production host: Escherichia coli (E. coli)
References: UniProt: S1IIE7, UniProt: P0A6G7*PLUS, endopeptidase Clp
#2: Protein
ATP-dependent Clp protease ATP-binding subunit ClpA


Mass: 84321.844 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: clpA, lopD, b0882, JW0866 / Production host: Escherichia coli (E. coli) / References: UniProt: P0ABH9
#3: Protein/peptide RepA, green fluorescent protein fusion


Mass: 2060.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
Has ligand of interestN

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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
1ClpAPCOMPLEX#1-#30RECOMBINANT
2ATP-dependent Clp protease ATP-binding subunit ClpACOMPLEX#11RECOMBINANT
3ATP-dependent Clp protease proteolytic subunitCOMPLEX#21RECOMBINANT
4RepA(1-25)-GFPCOMPLEX#31RECOMBINANT
Molecular weightValue: 0.84 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23Escherichia coli (E. coli)562
34synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli BL21(DE3) (bacteria)469008
23Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 / Nominal magnification: 105000 X / Calibrated magnification: 58616 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 69 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
Image scansWidth: 11520 / Height: 8184

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Processing

SoftwareName: PHENIX / Version: 1.17_3644: / Classification: refinement
EM softwareName: cryoSPARC / Version: 2 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 57848 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00349002
ELECTRON MICROSCOPYf_angle_d0.50666184
ELECTRON MICROSCOPYf_dihedral_angle_d7.6926764
ELECTRON MICROSCOPYf_chiral_restr0.047562
ELECTRON MICROSCOPYf_plane_restr0.0048558

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