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- EMDB-21198: ClpP1P2 complex from M. tuberculosis bound to ADEP -

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Basic information

Entry
Database: EMDB / ID: EMD-21198
TitleClpP1P2 complex from M. tuberculosis bound to ADEP
Map dataClpP1P2 complex from M. tuberculosis with ADEP
Sample
  • Complex: ClpP1P2 complex with ADEP
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit 1
  • Protein or peptide: R0M-WFP-ALO-PRO-YCP-ALA-MP8
KeywordsComplex / protease / ClpP / tuberculosis / HYDROLASE / HYDROLASE-ANTIBIOTIC complex
Function / homology
Function and homology information


endopeptidase Clp / endopeptidase Clp complex / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / peptidoglycan-based cell wall / ATPase binding / serine-type endopeptidase activity / plasma membrane / cytosol / cytoplasm
Similarity search - Function
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 / ClpP/crotonase-like domain superfamily
Similarity search - Domain/homology
ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease proteolytic subunit 2 / ATP-dependent Clp protease proteolytic subunit 1
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsRipstein ZA / Vahidi S
Funding support Canada, 2 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)FDN-503573 Canada
Canadian Institutes of Health Research (CIHR)PJT-162186 Canada
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: An allosteric switch regulates ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR.
Authors: Siavash Vahidi / Zev A Ripstein / Jordan B Juravsky / Enrico Rennella / Alfred L Goldberg / Anthony K Mittermaier / John L Rubinstein / Lewis E Kay /
Abstract: The 300-kDa ClpP1P2 protease from collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other ...The 300-kDa ClpP1P2 protease from collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all of the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of antituberculosis (anti-TB) therapeutics that target the Clp system. Here, we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Electron cryomicroscopy (cryo-EM) structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key β-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that, on binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function and shows extensive allosteric interactions involving both intra- and interring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target .
History
DepositionJan 8, 2020-
Header (metadata) releaseMar 18, 2020-
Map releaseMar 18, 2020-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vgn
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6vgn
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21198.png

Downloads & links

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Map

FileDownload / File: emd_21198.map.gz / Format: CCP4 / Size: 23.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClpP1P2 complex from M. tuberculosis with ADEP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 184 pix.
= 195.04 Å		1.06 Å/pix.
x 184 pix.
= 195.04 Å		1.06 Å/pix.
x 184 pix.
= 195.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.3 / Movie #1: 1.3
Minimum - Maximum-4.110243 - 6.395691
Average (Standard dev.)0.0137058 (±0.36020818)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions184184184
Spacing184184184
CellA=B=C: 195.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z184184184
origin x/y/z0.0000.0000.000
length x/y/z195.040195.040195.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS184184184
D min/max/mean-4.1106.3960.014

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Supplemental data

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Sample components

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Entire : ClpP1P2 complex with ADEP

EntireName: ClpP1P2 complex with ADEP
Components
  • Complex: ClpP1P2 complex with ADEP
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit 1
  • Protein or peptide: R0M-WFP-ALO-PRO-YCP-ALA-MP8

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Supramolecule #1: ClpP1P2 complex with ADEP

SupramoleculeName: ClpP1P2 complex with ADEP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Complex formed between P1 and P2 heptameric rings with bound ADEP
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: ATP-dependent Clp protease proteolytic subunit

MacromoleculeName: ATP-dependent Clp protease proteolytic subunit / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 21.914957 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: ILPSFIEHSS FGVKESNPYN KLFEERIIFL GVQVDDASAN DIMAQLLVLE SLDPDRDITM YINSPGGGFT SLMAIYDTMQ YVRADIQTV CLGQAASAAA VLLAAGTPGK RMALPNARVL IHQPSLSGVI QGQFSDLEIQ AAEIERMRTL METTLARHTG K DAGVIRKD ...String:
ILPSFIEHSS FGVKESNPYN KLFEERIIFL GVQVDDASAN DIMAQLLVLE SLDPDRDITM YINSPGGGFT SLMAIYDTMQ YVRADIQTV CLGQAASAAA VLLAAGTPGK RMALPNARVL IHQPSLSGVI QGQFSDLEIQ AAEIERMRTL METTLARHTG K DAGVIRKD TDRDKILTAE EAKDYGIIDT VLEYRKLSAQ TA

UniProtKB: ATP-dependent Clp protease proteolytic subunit

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Macromolecule #2: ATP-dependent Clp protease proteolytic subunit 1

MacromoleculeName: ATP-dependent Clp protease proteolytic subunit 1 / type: protein_or_peptide / ID: 2 / Number of copies: 7 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 21.065934 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MRSNSQGLSL TDSVYERLLS ERIIFLGSEV NDEIANRLCA QILLLAAEDA SKDISLYINS PGGSISAGMA IYDTMVLAPC DIATYAMGM AASMGEFLLA AGTKGKRYAL PHARILMHQP LGGVTGSAAD IAIQAEQFAV IKKEMFRLNA EFTGQPIERI E ADSDRDRW ...String:
MRSNSQGLSL TDSVYERLLS ERIIFLGSEV NDEIANRLCA QILLLAAEDA SKDISLYINS PGGSISAGMA IYDTMVLAPC DIATYAMGM AASMGEFLLA AGTKGKRYAL PHARILMHQP LGGVTGSAAD IAIQAEQFAV IKKEMFRLNA EFTGQPIERI E ADSDRDRW FTAAEALEYG FVDHIITRAH VNGEAQ

UniProtKB: ATP-dependent Clp protease proteolytic subunit 1

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Macromolecule #3: R0M-WFP-ALO-PRO-YCP-ALA-MP8

MacromoleculeName: R0M-WFP-ALO-PRO-YCP-ALA-MP8 / type: protein_or_peptide / ID: 3 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 800.888 Da
SequenceString:
(R0M)(WFP)(ALO)P(YCP)A(MP8)

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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

Concentration20 mg/mL
BufferpH: 7
Component:
ConcentrationName
50.0 mMImidazole
100.0 mMPotassium Chloride
5.0 mMDithiothreitol
0.025 %IGEPAL-CA630

Details: IGEPAL-CA630 was added shortly prior to vitrification
GridSupport film - topology: HOLEY / Support film - Film thickness: 30 / Details: unspecified
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III / Details: Blotted for 4.5 seconds at an offset of -5 mm.
DetailsMono-disperse complexes

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 70.0 K / Max: 77.0 K
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 725 / Average exposure time: 60.0 sec. / Average electron dose: 43.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 366129
Startup modelType of model: OTHER / Details: ab initio model in cryoSPARC
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.10) / Number images used: 192430
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.10)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 2.10)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 2.10)

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Atomic model buiding 1

Initial modelPDB ID:

5dzk


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6vgn:
ClpP1P2 complex from M. tuberculosis bound to ADEP

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