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- PDB-9c87: Cryo-EM Structure of a Proteolytic ClpXP AAA+ Machine Poised to U... -

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Basic information

Entry
Database: PDB / ID: 9c87
TitleCryo-EM Structure of a Proteolytic ClpXP AAA+ Machine Poised to Unfold a Linear-Degron DHFR-ssrA Substrate Bound with MTX
Components
  • (ATP-dependent Clp protease ...) x 2
  • Dihydrofolate reductase
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 / dihydrofolate metabolic process / protein quality control for misfolded or incompletely synthesized proteins / dihydrofolate reductase ...protein denaturation / HslUV protease complex / endopeptidase Clp / endopeptidase Clp complex / positive regulation of programmed cell death / response to temperature stimulus / ATP-dependent peptidase activity / dihydrofolate metabolic process / protein quality control for misfolded or incompletely synthesized proteins / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process / protein unfolding / proteasomal protein catabolic process / tetrahydrofolate biosynthetic process / serine-type peptidase activity / one-carbon metabolic process / proteolysis involved in protein catabolic process / ATP-dependent protein folding chaperone / response to radiation / disordered domain specific binding / unfolded protein binding / NADP 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 / Dihydrofolate reductase / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. / Dihydrofolate reductase (DHFR) domain profile. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase-like domain superfamily / 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 / METHOTREXATE / dihydrofolate reductase / 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: 3.7 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

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Assembly

Deposited unit
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
S: Dihydrofolate reductase
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)610,17432
Polymers606,81921
Non-polymers3,35511
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

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


Mass: 42412.863 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: clpX, lopC, b0438, JW0428 / Production host: Escherichia coli BL21 (bacteria) / 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 BL21 (bacteria) / References: UniProt: P0A6G7, endopeptidase Clp

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Protein , 1 types, 1 molecules S

#2: Protein Dihydrofolate reductase


Mass: 23777.590 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: folA, HmCmsJML131_03510 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A4C7B7M9, dihydrofolate reductase

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Non-polymers , 4 types, 11 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
#7: Chemical ChemComp-MTX / METHOTREXATE


Mass: 454.439 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H22N8O5 / Feature type: SUBJECT OF INVESTIGATION / Comment: chemotherapy*YM

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Details

Has ligand of interestY
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: ClpX.ClpP.Linear-Degron DHFR-ssrA / Type: COMPLEX / Entity ID: #1-#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 software
IDNameVersionCategory
7cryoSPARC4.4.1model fitting
13PHENIX1.14_3260:model refinement
CTF correctionDetails: Patch CTF estimation, cryoSPARC / Type: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71203 / Symmetry type: POINT

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