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- EMDB-23001: NmClpP compressed conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-23001
TitleNmClpP compressed conformation
Map dataNmClpP alone pH 7.0
Sample
  • Complex: caseinolytic protease
Biological speciesNeisseria meningitidis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsRipstein ZA / Vahidi S / Rubinstein JL / Kay LE
Funding support Canada, 3 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)FDN-503573 Canada
Canadian Institutes of Health Research (CIHR)PJT-148564 Canada
Canadian Institutes of Health Research (CIHR)PJT-162186 Canada
CitationJournal: J Am Chem Soc / Year: 2020
Title: A pH-Dependent Conformational Switch Controls ClpP Protease Function.
Authors: Zev A Ripstein / Siavash Vahidi / John L Rubinstein / Lewis E Kay /
Abstract: ClpPs are a conserved family of serine proteases that collaborate with ATP-dependent translocases to degrade protein substrates. Drugs targeting these enzymes have attracted interest for the ...ClpPs are a conserved family of serine proteases that collaborate with ATP-dependent translocases to degrade protein substrates. Drugs targeting these enzymes have attracted interest for the treatment of cancer and bacterial infections due to their critical role in mitochondrial and bacterial proteostasis, respectively. As such, there is significant interest in understanding structure-function relationships in this protein family. ClpPs are known to crystallize in extended, compact, and compressed forms; however, it is unclear what conditions favor the formation of each form and whether they are populated by wild-type enzymes in solution. Here, we use cryo-EM and solution NMR spectroscopy to demonstrate that a pH-dependent conformational switch controls an equilibrium between the active extended and inactive compressed forms of ClpP from the Gram-negative pathogen . Our findings provide insight into how ClpPs exploit their rugged energy landscapes to enable key conformational changes that regulate their function.
History
DepositionNov 18, 2020-
Header (metadata) releaseDec 23, 2020-
Map releaseDec 23, 2020-
UpdateDec 23, 2020-
Current statusDec 23, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23001.png

Downloads & links

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Map

FileDownload / File: emd_23001.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNmClpP alone pH 7.0
Voxel sizeX=Y=Z: 1.45 Å
Density
Contour LevelBy AUTHOR: 0.7 / Movie #1: 0.7
Minimum - Maximum-2.4524817 - 3.165143
Average (Standard dev.)0.006924257 (±0.17274721)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 232.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.451.451.45
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z232.000232.000232.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ180180180
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-2.4523.1650.007

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Supplemental data

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Sample components

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Entire : caseinolytic protease

EntireName: caseinolytic protease
Components
  • Complex: caseinolytic protease

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Supramolecule #1: caseinolytic protease

SupramoleculeName: caseinolytic protease / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Neisseria meningitidis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pet28a
Molecular weightTheoretical: 316 KDa

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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: (Name: Imidazole, Potassium Chloride)
GridModel: Homemade / Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 30.0 nm
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-30 / Number grids imaged: 1 / Average exposure time: 15.0 sec. / Average electron dose: 35.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 25000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 2.10)
Final reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.10) / Number images used: 68731
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.10)

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