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- PDB-9c88: Cryo-EM Structure of a Proteolytic ClpXP AAA+ Machine Translocati... -

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Basic information

Entry
Database: PDB / ID: 9c88
TitleCryo-EM Structure of a Proteolytic ClpXP AAA+ Machine Translocating a Portion of a Branched-Degron DHFR Substrate
Components
  • (ATP-dependent Clp protease ...) x 2
  • Branched-Degron DHFR
KeywordsCHAPERONE / ClpXP / full-engaged state / AAA protease
Function / homology
Function and homology information


protein denaturation / 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 ...protein denaturation / 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 / proteolysis involved in 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 / identical protein binding / membrane / cytosol
Similarity search - Function
Zinc finger, ClpX C4-type superfamily / ClpX C4-type zinc finger / Clp protease, ATP-binding subunit ClpX / Zinc finger, ClpX C4-type / Clp protease, ATP-binding subunit ClpX, bacteria / ClpX zinc binding (ZB) domain profile. / ClpX C4-type zinc finger / : / ClpP, Ser active site / Endopeptidase Clp serine active site. ...Zinc finger, ClpX C4-type superfamily / ClpX C4-type zinc finger / Clp protease, ATP-binding subunit ClpX / Zinc finger, ClpX C4-type / Clp protease, ATP-binding subunit ClpX, bacteria / 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 / 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 / ADENOSINE-5'-TRIPHOSPHATE / ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease ATP-binding subunit ClpX
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsGhanbarpour, A. / Sauer, R.T. / Davis, J.H.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)R01-GM144542 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM141517 United States
National Science Foundation (NSF, United States)2046778 United States
CitationJournal: Nat Commun / Year: 2024
Title: A proteolytic AAA+ machine poised to unfold protein substrates.
Authors: Alireza Ghanbarpour / Robert T Sauer / Joseph H Davis /
Abstract: AAA+ proteolytic machines unfold proteins before degrading them. Here, we present cryoEM structures of ClpXP-substrate complexes that reveal a postulated but heretofore unseen intermediate in ...AAA+ proteolytic machines unfold proteins before degrading them. Here, we present cryoEM structures of ClpXP-substrate complexes that reveal a postulated but heretofore unseen intermediate in substrate unfolding/degradation. A ClpX hexamer draws natively folded substrates tightly against its axial channel via interactions with a fused C-terminal degron tail and ClpX-RKH loops that flexibly conform to the globular substrate. The specific ClpX-substrate contacts observed vary depending on the substrate degron and affinity tags, helping to explain ClpXP's ability to unfold/degrade a wide array of different cellular substrates. Some ClpX contacts with native substrates are enabled by upward movement of the seam subunit in the AAA+ spiral, a motion coupled to a rearrangement of contacts between the ClpX unfoldase and ClpP peptidase. Our structures additionally highlight ClpX's ability to translocate a diverse array of substrate topologies, including the co-translocation of two polypeptide chains.
History
DepositionJun 12, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 16, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 30, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
S: Branched-Degron DHFR
A: ATP-dependent Clp protease ATP-binding subunit ClpX
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
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
u: ATP-dependent Clp protease proteolytic subunit
v: ATP-dependent Clp protease proteolytic subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)586,46831
Polymers583,56821
Non-polymers2,90010
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein/peptide , 1 types, 1 molecules S

#1: Protein/peptide Branched-Degron DHFR


Mass: 869.063 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Since the side chain density is not well-resolved for the portion of the substrate within the ClpX axial channel, they we modeled it as poly-UNK. Therefore, the alignment with the actual ...Details: Since the side chain density is not well-resolved for the portion of the substrate within the ClpX axial channel, they we modeled it as poly-UNK. Therefore, the alignment with the actual sequence of the substrate is not relevant.
Source: (synth.) Escherichia coli (E. coli)

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ATP-dependent Clp protease ... , 2 types, 20 molecules ABCDEFhijklmnpqrstuv

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


Mass: 42355.812 Da / Num. of mol.: 6 / Fragment: UNP residues 62-424
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: clpX, lopC, b0438, JW0428 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A6H1
#3: 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.: 14 / Fragment: UNP residues 16-207
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: clpP, lopP, b0437, JW0427 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A6G7, endopeptidase Clp

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Non-polymers , 3 types, 10 molecules

#4: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM

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Details

Has ligand of interestY
Has protein modificationN
Sequence detailsThe substrate density is not sufficient to determine which part of it can be visualized in the ...The substrate density is not sufficient to determine which part of it can be visualized in the structure. It was modeled as poly-UNK. The actual studied sequence is: MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLDKPVIMGRH TWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPE IMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFH DADAQNSHS YCFEILERRGSHLGLIEVEKPLYCVEP FVGETAHFEIELSEPDVHGQWKLTSHHHHHH.

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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.DHFR / Type: COMPLEX / Entity ID: #2-#3 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
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 defocus max: 1750 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 47.54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286: / Category: model refinement
CTF correctionDetails: Patch CTF estimation, cryoSPARC / Type: NONE
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 178399 / Symmetry type: POINT

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