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- EMDB-7952: Caseinolytic protease (ClpP) from Staphylococcus aureus mutant - V7A -

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Basic information

Entry
Database: EMDB / ID: EMD-7952
TitleCaseinolytic protease (ClpP) from Staphylococcus aureus mutant - V7A
Map dataprimary map
Sample
  • Complex: Caseinolytic protease from Staphylococcus aureus (V7A)Endopeptidase Clp
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
KeywordsProtease / HYDROLASE
Function / homology
Function and homology information


endopeptidase Clp / ATP-dependent peptidase activity / serine-type endopeptidase activity / 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
Similarity search - Component
Biological speciesStaphylococcus aureus (strain Newman) (bacteria) / Staphylococcus aureus subsp. aureus str. Newman (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsRipstein ZA / Vahidi S / Kay LE / Rubinstein JL
Funding support Canada, 6 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
Canadian Institutes of Health Research (CIHR) Canada
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
Canadian Institutes of Health Research (CIHR) Canada
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Reversible inhibition of the ClpP protease via an N-terminal conformational switch.
Authors: Siavash Vahidi / Zev A Ripstein / Massimiliano Bonomi / Tairan Yuwen / Mark F Mabanglo / Jordan B Juravsky / Kamran Rizzolo / Algirdas Velyvis / Walid A Houry / Michele Vendruscolo / John L ...Authors: Siavash Vahidi / Zev A Ripstein / Massimiliano Bonomi / Tairan Yuwen / Mark F Mabanglo / Jordan B Juravsky / Kamran Rizzolo / Algirdas Velyvis / Walid A Houry / Michele Vendruscolo / John L Rubinstein / Lewis E Kay /
Abstract: Protein homeostasis is critically important for cell viability. Key to this process is the refolding of misfolded or aggregated proteins by molecular chaperones or, alternatively, their degradation ...Protein homeostasis is critically important for cell viability. Key to this process is the refolding of misfolded or aggregated proteins by molecular chaperones or, alternatively, their degradation by proteases. In most prokaryotes and in chloroplasts and mitochondria, protein degradation is performed by the caseinolytic protease ClpP, a tetradecamer barrel-like proteolytic complex. Dysregulating ClpP function has shown promise in fighting antibiotic resistance and as a potential therapy for acute myeloid leukemia. Here we use methyl-transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, biochemical assays, and molecular dynamics simulations to characterize the structural dynamics of ClpP from (SaClpP) in wild-type and mutant forms in an effort to discover conformational hotspots that regulate its function. Wild-type SaClpP was found exclusively in the active extended form, with the N-terminal domains of its component protomers in predominantly β-hairpin conformations that are less well-defined than other regions of the protein. A hydrophobic site was identified that, upon mutation, leads to unfolding of the N-terminal domains, loss of SaClpP activity, and formation of a previously unobserved split-ring conformation with a pair of 20-Å-wide pores in the side of the complex. The extended form of the structure and partial activity can be restored via binding of ADEP small-molecule activators. The observed structural plasticity of the N-terminal gates is shown to be a conserved feature through studies of and ClpP, suggesting a potential avenue for the development of molecules to allosterically modulate the function of ClpP.
History
DepositionMay 29, 2018-
Header (metadata) releaseJun 13, 2018-
Map releaseJun 27, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.54
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.54
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6dkf
  • Surface level: 0.54
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7952.png

Downloads & links

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Map

FileDownload / File: emd_7952.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationprimary map
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.54 / Movie #1: 0.54
Minimum - Maximum-2.3748946 - 3.3238964
Average (Standard dev.)0.010922023 (±0.13092919)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 203.51999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z203.520203.520203.520
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ384384384
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-2.3753.3240.011

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Supplemental data

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Sample components

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Entire : Caseinolytic protease from Staphylococcus aureus (V7A)

EntireName: Caseinolytic protease from Staphylococcus aureus (V7A)Endopeptidase Clp
Components
  • Complex: Caseinolytic protease from Staphylococcus aureus (V7A)Endopeptidase Clp
    • Protein or peptide: ATP-dependent Clp protease proteolytic subunit

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Supramolecule #1: Caseinolytic protease from Staphylococcus aureus (V7A)

SupramoleculeName: Caseinolytic protease from Staphylococcus aureus (V7A)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Staphylococcus aureus (strain Newman) (bacteria) / Strain: Newman
Molecular weightTheoretical: 301 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: 14 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Staphylococcus aureus subsp. aureus str. Newman (bacteria)
Strain: Newman
Molecular weightTheoretical: 21.508479 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNLIPTAIET TNRGERAYDI YSRLLKDRII MLGSQIDDNV ANSIVSQLLF LQAQDSEKDI YLYINSPGGS VTAGFAIYDT IQHIKPDVQ TICIGMAASM GSFLLAAGAK GKRFALPNAE VMIHQPLGGA QGQATEIEIA ANHILKTREK LNRILSERTG Q SIEKIQKD ...String:
MNLIPTAIET TNRGERAYDI YSRLLKDRII MLGSQIDDNV ANSIVSQLLF LQAQDSEKDI YLYINSPGGS VTAGFAIYDT IQHIKPDVQ TICIGMAASM GSFLLAAGAK GKRFALPNAE VMIHQPLGGA QGQATEIEIA ANHILKTREK LNRILSERTG Q SIEKIQKD TDRDNFLTAE EAKEYGLIDE VMVPETK

UniProtKB: ATP-dependent Clp protease proteolytic subunit

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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

Concentration30 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
100.0 mMKClpotassium chloride
1.0 mMEDTAEthylenediaminetetraacetic acidethylenediaminetetraacetic acid
5.0 mMMgCl2magnesium chloride
5.0 %Igepal CA-630
50.0 mMimidazole
GridSupport film - topology: HOLEY / Support film - Film thickness: 35 / Details: unspecified
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III / Details: Modified Vitrobot.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 3.2 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Frames/image: 1-30 / Number grids imaged: 2 / Number real images: 1837 / Average exposure time: 60.0 sec. / Average electron dose: 43.0 e/Å2 / Details: movies were collected with 44 fractions
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 878240
Startup modelType of model: INSILICO MODEL / Details: Ab initio algorithm in cryoSPARC
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 0.5)
Final 3D classificationNumber classes: 5 / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 0.5)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 0.5) / Number images used: 324522

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Atomic model buiding 1

Initial model
PDB IDChain

3v5e

chain_id: A, residue_range: 20-193, source_name: PDB, initial_model_type: experimental model

3qwd

chain_id: A, residue_range: 20-193, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6dkf:
Caseinolytic protease (ClpP) from Staphylococcus aureus mutant - V7A

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