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- PDB-6wrf: ClpX-ClpP complex bound to GFP-ssrA, recognition complex -

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Basic information

Entry
Database: PDB / ID: 6wrf
TitleClpX-ClpP complex bound to GFP-ssrA, recognition complex
Components
  • (ATP-dependent Clp protease ...) x 2
  • Green fluorescent protein
KeywordsCHAPERONE / Protein degradation / AAA+ protease complex
Function / homology
Function and homology information


protein denaturation / HslUV protease complex / endopeptidase Clp complex / endopeptidase Clp / 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 / serine-type peptidase activity ...protein denaturation / HslUV protease complex / endopeptidase Clp complex / endopeptidase Clp / 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 / serine-type peptidase activity / proteolysis involved in protein catabolic process / bioluminescence / generation of precursor metabolites and energy / proteasomal protein catabolic process / ATP-dependent protein folding chaperone / response to radiation / disordered domain specific binding / unfolded protein binding / ATPase binding / response to heat / protease binding / protein dimerization activity / cell division / serine-type endopeptidase activity / ATP hydrolysis activity / proteolysis / zinc ion binding / ATP binding / membrane / identical protein binding / cytosol
Similarity search - Function
Clp protease, ATP-binding subunit ClpX, bacteria / Zinc finger, ClpX C4-type superfamily / ClpX C4-type zinc finger / Clp protease, ATP-binding subunit ClpX / Zinc finger, ClpX C4-type / ClpX zinc binding (ZB) domain profile. / ClpX C4-type zinc finger / ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpP, histidine active site ...Clp protease, ATP-binding subunit ClpX, bacteria / Zinc finger, ClpX C4-type superfamily / ClpX C4-type zinc finger / Clp protease, ATP-binding subunit ClpX / Zinc finger, ClpX C4-type / ClpX zinc binding (ZB) domain profile. / ClpX C4-type zinc finger / ClpP, Ser active site / Endopeptidase Clp serine active site. / 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 / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / ClpP/crotonase-like domain superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / 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 / ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease ATP-binding subunit ClpX / Green fluorescent protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Aequorea victoria (jellyfish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsFei, X. / Sauer, R.T.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM-101988 United States
CitationJournal: Elife / Year: 2020
Title: Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates.
Authors: Xue Fei / Tristan A Bell / Sarah R Barkow / Tania A Baker / Robert T Sauer /
Abstract: When ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity. In and other eubacteria, the ...When ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity. In and other eubacteria, the tmRNA system rescues stalled ribosomes and adds an ssrA tag or degron to the C-terminus of the incomplete protein, which directs degradation by the AAA+ ClpXP protease. Here, we present cryo-EM structures of ClpXP bound to the ssrA degron. C-terminal residues of the ssrA degron initially bind in the top of an otherwise closed ClpX axial channel and subsequently move deeper into an open channel. For short-degron protein substrates, we show that unfolding can occur directly from the initial closed-channel complex. For longer degron substrates, our studies illuminate how ClpXP transitions from specific recognition into a nonspecific unfolding and translocation machine. Many AAA+ proteases and protein-remodeling motors are likely to employ similar multistep recognition and engagement strategies.
History
DepositionApr 29, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 4, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 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

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Structure visualization

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Assembly

Deposited unit
B: ATP-dependent Clp protease ATP-binding subunit ClpX
C: ATP-dependent Clp protease ATP-binding subunit ClpX
D: ATP-dependent Clp protease ATP-binding subunit ClpX
E: ATP-dependent Clp protease ATP-binding subunit ClpX
F: ATP-dependent Clp protease ATP-binding subunit ClpX
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
S: Green fluorescent protein
A: ATP-dependent Clp protease ATP-binding subunit ClpX
hetero molecules


Theoretical massNumber of molelcules
Total (without water)451,92425
Polymers448,75914
Non-polymers3,16511
Water1267
1


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

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Components

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ATP-dependent Clp protease ... , 2 types, 13 molecules BCDEFAHIJKLMN

#1: Protein
ATP-dependent Clp protease ATP-binding subunit ClpX / ATP-dependent unfoldase ClpX


Mass: 42355.812 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: clpX, lopC, b0438, JW0428 / Plasmid: pacyc / Production host: Escherichia coli (E. coli) / Strain (production host): ER2566 / References: UniProt: P0A6H1
#2: Protein
ATP-dependent Clp protease proteolytic subunit / / Caseinolytic protease / Endopeptidase Clp / Heat shock protein F21.5 / Protease Ti


Mass: 23468.869 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: clpP, lopP, b0437, JW0427 / Production host: Escherichia coli (E. coli) / Strain (production host): ER2566 / References: UniProt: P0A6G7, endopeptidase Clp

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Protein , 1 types, 1 molecules S

#3: Protein Green fluorescent protein /


Mass: 30341.744 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aequorea victoria (jellyfish) / Gene: GFP / Plasmid: pet / Production host: Escherichia coli (E. coli) / Strain (production host): ER2566 / References: UniProt: P42212

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Non-polymers , 4 types, 18 molecules

#4: 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.: 5 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
#6: 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
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Formula: H2O

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Details

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

ComponentName: ClpX-ClpP-GFPssrA-ATP/ATPrS complex during target recognition
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: ER2566
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
225 mMMgCl21
3100 mMKCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: rapid mixing of ClpX, ClpP, ATPrS with GFPssrA substrate and ATP.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 X / Nominal defocus max: -2500 nm / Nominal defocus min: -800 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 54 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4525

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.0.8particle selection
2SerialEMimage acquisition
4CTFFIND4.1.12CTF correction
7UCSF Chimeramodel fitting
9PHENIX1.14model refinement
10RELION3.0.8initial Euler assignment
11RELION3.0.8final Euler assignment
12RELION3.0.8classification
13RELION3.0.83D reconstruction
14PHENIX1.143D reconstructionmap sharpening
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 139817 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6PO1

6po1


Accession code: 6PO1 / Source name: PDB / Type: experimental model

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