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- PDB-6uqo: ClpA/ClpP Engaged State bound to RepA-GFP -

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Basic information

Entry
Database: PDB / ID: 6uqo
TitleClpA/ClpP Engaged State bound to RepA-GFP
Components
  • ATP-dependent Clp endopeptidase proteolytic subunit ClpP
  • ATP-dependent Clp protease ATP-binding subunit ClpA
  • RepA-GFP
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 / cytosol / cytoplasm
Similarity search - Function
ATP-dependent Clp protease ATP-binding subunit ClpA / 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 / ClpP, Ser active site / Endopeptidase Clp serine active site. ...ATP-dependent Clp protease ATP-binding subunit ClpA / 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 / ClpP, Ser active site / Endopeptidase Clp serine active site. / : / Clp, repeat (R) domain / Clp repeat (R) domain profile. / ClpP, histidine active site / Endopeptidase Clp histidine active site. / Clp, N-terminal domain superfamily / ATP-dependent Clp protease proteolytic subunit / Clp protease proteolytic subunit /Translocation-enhancing protein TepA / Clp protease / ClpA/B family / Helicase, Ruva Protein; domain 3 - #60 / Clp ATPase, C-terminal / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / AAA domain (Cdc48 subfamily) / 2-enoyl-CoA Hydratase; Chain A, domain 1 / 2-enoyl-CoA Hydratase; Chain A, domain 1 / ClpP/crotonase-like domain superfamily / Helicase, Ruva Protein; domain 3 / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Alpha-Beta Complex / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-dependent Clp protease ATP-binding subunit ClpA / ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease ATP-binding subunit ClpA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
unidentified (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLopez, K.L. / Rizo, A.N. / 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
DepositionOct 21, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 22, 2020Provider: repository / Type: Initial release
Revision 1.1May 6, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2May 27, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 16, 2022Group: Author supporting evidence / Database references / Category: database_2 / pdbx_audit_support
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization
Revision 1.4Dec 25, 2024Group: Advisory / Data collection ...Advisory / Data collection / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_validate_close_contact / struct_conn
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
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
G: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
H: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
I: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
J: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
K: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
L: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
M: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
N: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
O: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
P: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
Q: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
R: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
S: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
T: ATP-dependent Clp endopeptidase proteolytic subunit ClpP
X: RepA-GFP
Y: RepA-GFP
hetero molecules


Theoretical massNumber of molelcules
Total (without water)693,21234
Polymers687,41422
Non-polymers5,79912
Water00
1


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

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Components

#1: Protein
ATP-dependent Clp protease ATP-binding subunit ClpA


Mass: 64204.504 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, FAZ83_15050 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A4S4P650, UniProt: P0ABH9*PLUS, endopeptidase Clp
#2: Protein
ATP-dependent Clp endopeptidase proteolytic subunit ClpP


Mass: 21472.762 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: clpP, FAZ83_16765 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A4V3YU15, UniProt: P0A6G7*PLUS, endopeptidase Clp
#3: Protein/peptide RepA-GFP


Mass: 783.958 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: 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.: 7 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
Has ligand of interestN
Has protein modificationN

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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: ClpAP / Type: COMPLEX / Entity ID: #2-#3 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97000 / Symmetry type: POINT

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