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- EMDB-15241: Mycobacterium tuberculosis ClpC1 hexamer structure bound to the n... -

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Basic information

Entry
Database: EMDB / ID: EMD-15241
TitleMycobacterium tuberculosis ClpC1 hexamer structure bound to the natural product antibiotic Cyclomarin
Map dataSharpened map of Mycobacterium tuberculosis ClpC1 cyclomarin
Sample
  • Complex: Mycobacterium tuberculosis ClpC1 in complex with the natural product antibiotic Cyclomarin
    • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpC1
  • Protein or peptide: Bound polypeptide
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Function / homology
Function and homology information


protein folding chaperone / peptidoglycan-based cell wall / cellular response to heat / protein homodimerization activity / ATP hydrolysis activity / ATP binding / plasma membrane / cytosol
Similarity search - Function
UVR domain / UVR domain profile. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp amino terminal domain, pathogenicity island component / Clp repeat (R) domain profile. / Clp, repeat (R) domain / Clp, N-terminal domain superfamily ...UVR domain / UVR domain profile. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp amino terminal domain, pathogenicity island component / Clp repeat (R) domain profile. / Clp, repeat (R) domain / Clp, N-terminal domain superfamily / ClpA/B family / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / ATPase family associated with various cellular activities (AAA) / 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
ATP-dependent Clp protease ATP-binding subunit ClpC1
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.34 Å
AuthorsFelix J / Fraga H / Gragera M / Bueno T / Weinhaeupl K
Funding supportEuropean Union, 3 items
OrganizationGrant numberCountry
Other governmentiNEXT Discovery (871037)European Union
Other governmentCRIOMECORR project (ESFRI-2019-01-CSIC-16)European Union
Other governmentInstruct-ERIC (PID 15689)European Union
CitationJournal: J Biol Chem / Year: 2022
Title: Structure of the drug target ClpC1 unfoldase in action provides insights on antibiotic mechanism of action.
Authors: Katharina Weinhäupl / Marcos Gragera / M Teresa Bueno-Carrasco / Rocío Arranz / Olga Krandor / Tatos Akopian / Raquel Soares / Eric Rubin / Jan Felix / Hugo Fraga /
Abstract: The unfoldase ClpC1 is one of the most exciting drug targets against tuberculosis. This AAA+ unfoldase works in cooperation with the ClpP1P2 protease and is the target of at least four natural ...The unfoldase ClpC1 is one of the most exciting drug targets against tuberculosis. This AAA+ unfoldase works in cooperation with the ClpP1P2 protease and is the target of at least four natural product antibiotics: cyclomarin, ecumicin, lassomycin, and rufomycin. Although these molecules are promising starting points for drug development, their mechanisms of action remain largely unknown. Taking advantage of a middle domain mutant, we determined the first structure of Mycobacterium tuberculosis ClpC1 in its apo, cyclomarin-, and ecumicin-bound states via cryo-EM. The obtained structure displays features observed in other members of the AAA+ family and provides a map for further drug development. While the apo and cyclomarin-bound structures are indistinguishable and have N-terminal domains that are invisible in their respective EM maps, around half of the ecumicin-bound ClpC1 particles display three of their six N-terminal domains in an extended conformation. Our structural observations suggest a mechanism where ecumicin functions by mimicking substrate binding, leading to ATPase activation and changes in protein degradation profile.
History
DepositionJun 24, 2022-
Header (metadata) releaseOct 26, 2022-
Map releaseOct 26, 2022-
UpdateNov 23, 2022-
Current statusNov 23, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15241.png

Downloads & links

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Map

FileDownload / File: emd_15241.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of Mycobacterium tuberculosis ClpC1 cyclomarin
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 500 pix.
= 427.5 Å		0.86 Å/pix.
x 500 pix.
= 427.5 Å		0.86 Å/pix.
x 500 pix.
= 427.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.855 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum-2.2712488 - 4.016456
Average (Standard dev.)-0.0012224399 (±0.07257099)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 427.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15241_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: DeepEMhancer sharpened map of Mycobacterium tuberculosis ClpC1 cyclomarin...

Fileemd_15241_additional_1.map
AnnotationDeepEMhancer sharpened map of Mycobacterium tuberculosis ClpC1 cyclomarin
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15241_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15241_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Mycobacterium tuberculosis ClpC1 in complex with the natural prod...

EntireName: Mycobacterium tuberculosis ClpC1 in complex with the natural product antibiotic Cyclomarin
Components
  • Complex: Mycobacterium tuberculosis ClpC1 in complex with the natural product antibiotic Cyclomarin
    • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpC1
  • Protein or peptide: Bound polypeptide
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Mycobacterium tuberculosis ClpC1 in complex with the natural prod...

SupramoleculeName: Mycobacterium tuberculosis ClpC1 in complex with the natural product antibiotic Cyclomarin
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 567 KDa

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Macromolecule #1: ATP-dependent Clp protease ATP-binding subunit ClpC1

MacromoleculeName: ATP-dependent Clp protease ATP-binding subunit ClpC1 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: Hydrolases; Acting on peptide bonds (peptidases)
Source (natural)Organism: Mycobacterium tuberculosis (bacteria) / Strain: ATCC 25618 / H37Rv
Molecular weightTheoretical: 94.758695 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFERFTDRAR RVVVLAQEEA RMLNHNYIGT EHILLGLIHE GEGVAAKSLE SLGISLEGVR SQVEEIIGQG QQAPSGHIPF TPRAKKVLE LSLREALQLG HNYIGTEHIL LGLIREGEGV AAQVLVKLGA ELTRVRQQVI QLLSGYQGKE AAEAGTGGRG G ESGSPSTS ...String:
MFERFTDRAR RVVVLAQEEA RMLNHNYIGT EHILLGLIHE GEGVAAKSLE SLGISLEGVR SQVEEIIGQG QQAPSGHIPF TPRAKKVLE LSLREALQLG HNYIGTEHIL LGLIREGEGV AAQVLVKLGA ELTRVRQQVI QLLSGYQGKE AAEAGTGGRG G ESGSPSTS LVLDQFGRNL TAAAMEGKLD PVIGREKEIE RVMQVLSRRT KNNPVLIGEP GVGKTAVVEG LAQAIVHGEV PE TLKDKQL YTLDLGSLVA GSRYRGDFEE RLKKVLKEIN TRGDIILFID ELHTLVGAGA AEGAIDAASI LKPKLARGEL QTI GATTLD EYRKYIEKDA ALERRFQPVQ VGEPTVEHTI EILKGLRDRY EAHHRVSITD AAMVAAATLA DRYINDRFLP DKAI DLIDE AGARMRIRRM TAPPDLREFD EKIAEARREK ESAIDAQDFE KAASLRDREK TLVAQRAERE KQWRSGDLDV VAEVD DEQI AEVLGNWTGI PVFKLTEAET TRLLRMEEEL HKRIIGQEDA VKAVSKAIRR TRAGLKDPKR PSGSFIFAGP SGVGKT ELS KALANFLFGD DDALIQIDMG EFHDRFTASR LFGAPPGYVG YEEGGQLTEK VRRKPFSVVL FDEIEKAHQE IYNSLLQ VL EDGRLTDGQG RTVDFKNTVL IFTSNLGTSD ISKPVGLGFS KGGGENDYER MKQKVNDELK KHFRPEFLNR IDDIIVFH Q LTREEIIRMV DLMISRVAGQ LKSKDMALVL TDAAKALLAK RGFDPVLGAR PLRRTIQREI EDQLSEKILF EEVGPGQVV TVDVDNWDGE GPGEDAVFTF TGTRKPPAEP DLAKAGAHSA GGPEPAARLE HHHHHH

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Macromolecule #2: Bound polypeptide

MacromoleculeName: Bound polypeptide / type: protein_or_peptide / ID: 2 / Details: The actual sequence of the polypeptide is unknown. / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 1.975426 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 10 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

Concentration1 mg/mL
BufferpH: 7.4
Details: Hepes pH 7.4 50 mM, NaCl 100 mM, 10 mM MgCl2, ATP 1mM
VitrificationCryogen name: ETHANE
Details10 microM ClpC1 with 30 microM Cyclomarin

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average exposure time: 30.0 sec. / Average electron dose: 36.9 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: OTHER / Nominal defocus max: 3.1 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. v3.3.2)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.2) / Number images used: 102018
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.3.2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. v3.3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7abr

Output model

PDB-8a8v:
Mycobacterium tuberculosis ClpC1 hexamer structure bound to the natural product antibiotic Cyclomarin

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