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- EMDB-45299: Cryo-EM Structure of a Proteolytic ClpXP AAA+ Machine Poised to U... -

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Entry
Database: EMDB / ID: EMD-45299
TitleCryo-EM Structure of a Proteolytic ClpXP AAA+ Machine Poised to Unfold a Linear-Degron DHFR-ssrA Substrate Bound with MTX
Map data
Sample
  • Complex: ClpX.ClpP.Linear-Degron DHFR-ssrA
    • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpX
    • Protein or peptide: Dihydrofolate reductase
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: METHOTREXATE
KeywordsClpXP / full-engaged state / AAA protease / CHAPERONE
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
dihydrofolate reductase / ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease ATP-binding subunit ClpX
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsGhanbarpour A / Sauer RT / Davis JH
Funding support United States, 3 items
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, 2024-
Header (metadata) releaseOct 16, 2024-
Map releaseOct 16, 2024-
UpdateApr 30, 2025-
Current statusApr 30, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45299.png

Downloads & links

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Map

FileDownload / File: emd_45299.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.12 Å/pix.
x 256 pix.
= 286.003 Å		1.12 Å/pix.
x 256 pix.
= 286.003 Å		1.12 Å/pix.
x 256 pix.
= 286.003 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1172 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0676
Minimum - Maximum-0.18527427 - 0.41675952
Average (Standard dev.)0.0013106613 (±0.01885602)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 286.0032 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45299_msk_1.map
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Half map: #2

Fileemd_45299_half_map_1.map
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Half map: #1

Fileemd_45299_half_map_2.map
Projections & Slices
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Sample components

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Entire : ClpX.ClpP.Linear-Degron DHFR-ssrA

EntireName: ClpX.ClpP.Linear-Degron DHFR-ssrA
Components
  • Complex: ClpX.ClpP.Linear-Degron DHFR-ssrA
    • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpX
    • Protein or peptide: Dihydrofolate reductase
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: METHOTREXATE

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Supramolecule #1: ClpX.ClpP.Linear-Degron DHFR-ssrA

SupramoleculeName: ClpX.ClpP.Linear-Degron DHFR-ssrA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: ATP-dependent Clp protease ATP-binding subunit ClpX

MacromoleculeName: ATP-dependent Clp protease ATP-binding subunit ClpX / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 42.412863 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MGSSHHHHHH DYDIPTTENL YFQGSSALPT PHEIRNHLDD YVIGQEQAKK VLAVAVYNHY KRLRNGDTSN GVELGKSNIL LIGPTGSGK TLLAETLARL LDVPFTMADA TTLTEAGYVG EDVENIIQKL LQKSDYDVQK AQRGIVYIDE IDKISRKSDN P SITRDVSG ...String:
MGSSHHHHHH DYDIPTTENL YFQGSSALPT PHEIRNHLDD YVIGQEQAKK VLAVAVYNHY KRLRNGDTSN GVELGKSNIL LIGPTGSGK TLLAETLARL LDVPFTMADA TTLTEAGYVG EDVENIIQKL LQKSDYDVQK AQRGIVYIDE IDKISRKSDN P SITRDVSG EGVQQALLKL IEGTVAAVPP QGGRKHPQQE FLQVDTSKIL FICGGAFAGL DKVISHRVET GSGIGFGATV KA KSDKASE GELLAQVEPE DLIKFGLIPE FIGRLPVVAT LNELSEEALI QILKEPKNAL TKQYQALFNL EGVDLEFRDE ALD AIAKKA MARKTGARGL RSIVEAALLD TMYDLPSMED VEKVVIDESV IDGQSEPLLI YGKPEAQQAS GEG

UniProtKB: ATP-dependent Clp protease ATP-binding subunit ClpX

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Macromolecule #2: Dihydrofolate reductase

MacromoleculeName: Dihydrofolate reductase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: dihydrofolate reductase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 23.77759 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHHHHHHENL YFQGISLIAA LAVDRVIGME NAMPWNLPAD LAWFKRNTLD KPVIMGRHTW ESIGRPLPGR KNIILSSQPG TDDRVTWVK SVDEAIAACG DVPEIMVIGG GRVYEQFLPK AQKLYLTHID AEVEGDTHFP DYEPDDWESV FSEFHDADAQ N SHSYCFEI ...String:
MHHHHHHENL YFQGISLIAA LAVDRVIGME NAMPWNLPAD LAWFKRNTLD KPVIMGRHTW ESIGRPLPGR KNIILSSQPG TDDRVTWVK SVDEAIAACG DVPEIMVIGG GRVYEQFLPK AQKLYLTHID AEVEGDTHFP DYEPDDWESV FSEFHDADAQ N SHSYCFEI LERRGSHLGL IEVEKPLYCG SGSWSHPQFE KAANDENYAL AA

UniProtKB: dihydrofolate reductase

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Macromolecule #3: ATP-dependent Clp protease proteolytic subunit

MacromoleculeName: ATP-dependent Clp protease proteolytic subunit / type: protein_or_peptide / ID: 3 / Number of copies: 14 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 23.468869 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: LVPMVIEQTS RGERSFDIYS RLLKERVIFL TGQVEDHMAN LIVAQMLFLE AENPEKDIYL YINSPGGVIT AGMSIYDTMQ FIKPDVSTI CMGQAASMGA FLLTAGAKGK RFCLPNSRVM IHQPLGGYQG QATDIEIHAR EILKVKGRMN ELMALHTGQS L EQIERDTE ...String:
LVPMVIEQTS RGERSFDIYS RLLKERVIFL TGQVEDHMAN LIVAQMLFLE AENPEKDIYL YINSPGGVIT AGMSIYDTMQ FIKPDVSTI CMGQAASMGA FLLTAGAKGK RFCLPNSRVM IHQPLGGYQG QATDIEIHAR EILKVKGRMN ELMALHTGQS L EQIERDTE RDRFLSAPEA VEYGLVDSIL THRNENLYFQ SLEHHHHHH

UniProtKB: ATP-dependent Clp protease proteolytic subunit

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 3 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 3 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #7: METHOTREXATE

MacromoleculeName: METHOTREXATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: MTX
Molecular weightTheoretical: 454.439 Da
Chemical component information

ChemComp-MTX:
METHOTREXATE / chemotherapy*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 47.54 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.75 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 71203
Initial angle assignmentType: PROJECTION MATCHING / Details: Ab-initio reconstruction, cryoSPARC
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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Atomic model buiding 1

SoftwareName: cryoSPARC (ver. 4.4.1)
Output model

PDB-9c87:
Cryo-EM Structure of a Proteolytic ClpXP AAA+ Machine Poised to Unfold a Linear-Degron DHFR-ssrA Substrate Bound with MTX

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