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Yorodumi- EMDB-7951: cryo-EM density map of ClpP from Staphylococcus aureus with bound... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-7951 | |||||||||
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Title | cryo-EM density map of ClpP from Staphylococcus aureus with bound Acyldepsipeptide (V7A mutant) | |||||||||
Map data | ||||||||||
Sample |
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Biological species | Staphylococcus aureus (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 5.6 Å | |||||||||
Authors | Ripstein ZA / Vahidi S / Kay LE / Rubinstein JL | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_7951.map.gz | 14.7 MB | EMDB map data format | |
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Header (meta data) | emd-7951-v30.xml emd-7951.xml | 14.5 KB 14.5 KB | Display Display | EMDB header |
Images | emd_7951.png | 67.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-7951 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-7951 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_7951.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 1.45 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Caseinolytic protease from Staphylococcus aureus
Entire | Name: Caseinolytic protease from Staphylococcus aureusEndopeptidase Clp |
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Components |
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-Supramolecule #1: Caseinolytic protease from Staphylococcus aureus
Supramolecule | Name: Caseinolytic protease from Staphylococcus aureus / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Staphylococcus aureus (bacteria) / Strain: Newman |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) CodonPlus / Recombinant plasmid: pET24 |
Molecular weight | Experimental: 301 KDa |
-Macromolecule #1: Caseinolytic protease from Staphylococcus aureus
Macromolecule | Name: Caseinolytic protease from Staphylococcus aureus / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: endopeptidase Clp |
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Source (natural) | Organism: Staphylococcus aureus (bacteria) / Strain: Newman |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MNLIPTAIET TNRGERAYDI YSRLLKDRII MLGSQIDDNV ANSIVSQLLF LQAQDSEKDI YLYINSPGGS VTAGFAIYDT IQHIKPDVQT ICIGMAASMG SFLLAAGAKG KRFALPNAEV MIHQPLGGAQ GQATEIEIAA NHILKTREKL NRILSERTGQ SIEKIQKDTD ...String: MNLIPTAIET TNRGERAYDI YSRLLKDRII MLGSQIDDNV ANSIVSQLLF LQAQDSEKDI YLYINSPGGS VTAGFAIYDT IQHIKPDVQT ICIGMAASMG SFLLAAGAKG KRFALPNAEV MIHQPLGGAQ GQATEIEIAA NHILKTREKL NRILSERTGQ SIEKIQKDTD RDNFLTAEEA KEYGLIDEVM VPETK |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 30 mg/mL | ||||||||||||||||||
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Buffer | pH: 7 Component:
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Grid | Support film - topology: HOLEY / Support film - Film thickness: 35.0 nm / Details: unspecified | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III / Details: Modified Vitrobot. |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated defocus max: 4.2 µm / Calibrated defocus min: 1.2 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal magnification: 25000 |
Sample stage | Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-30 / Number grids imaged: 1 / Number real images: 75 / Average exposure time: 15.0 sec. / Average electron dose: 35.0 e/Å2 |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Particle selection | Number selected: 76614 |
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CTF correction | Software - Name: CTFFIND (ver. 4) |
Startup model | Type of model: INSILICO MODEL / Details: Ab initio algorithm in cryoSPARC |
Initial angle assignment | Type: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 0.5) |
Final 3D classification | Number classes: 5 / Software - Name: cryoSPARC |
Final angle assignment | Type: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 0.5) |
Final reconstruction | Number classes used: 1 / Applied symmetry - Point group: D7 (2x7 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 0.5) / Number images used: 37133 |