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- EMDB-21195: ClpXP from N. meningitidis- D7 symmetry applied -

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Basic information

Entry
Database: EMDB / ID: EMD-21195
TitleClpXP from N. meningitidis- D7 symmetry applied
Map dataClpXP complex from N. meningitidis, with D7 symmetry
Sample
  • Complex: ClpXP complex
Biological speciesNeisseria meningitidis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsRipstein ZA / Vahidi S / Kay LE / Rubinstein JL
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: Elife / Year: 2020
Title: A processive rotary mechanism couples substrate unfolding and proteolysis in the ClpXP degradation machinery.
Authors: Zev A Ripstein / Siavash Vahidi / Walid A Houry / John L Rubinstein / Lewis E Kay /
Abstract: The ClpXP degradation machine consists of a hexameric AAA+ unfoldase (ClpX) and a pair of heptameric serine protease rings (ClpP) that unfold, translocate, and subsequently degrade client proteins. ...The ClpXP degradation machine consists of a hexameric AAA+ unfoldase (ClpX) and a pair of heptameric serine protease rings (ClpP) that unfold, translocate, and subsequently degrade client proteins. ClpXP is an important target for drug development against infectious diseases. Although structures are available for isolated ClpX and ClpP rings, it remains unknown how symmetry mismatched ClpX and ClpP work in tandem for processive substrate translocation into the ClpP proteolytic chamber. Here, we present cryo-EM structures of the substrate-bound ClpXP complex from at 2.3 to 3.3 Å resolution. The structures allow development of a model in which the sequential hydrolysis of ATP is coupled to motions of ClpX loops that lead to directional substrate translocation and ClpX rotation relative to ClpP. Our data add to the growing body of evidence that AAA+ molecular machines generate translocating forces by a common mechanism.
History
DepositionJan 6, 2020-
Header (metadata) releaseJan 22, 2020-
Map releaseJan 22, 2020-
UpdateJan 22, 2020-
Current statusJan 22, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21195.png

Downloads & links

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Map

FileDownload / File: emd_21195.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClpXP complex from N. meningitidis, with D7 symmetry
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-5.201427 - 8.077001
Average (Standard dev.)0.0013097222 (±0.23969544)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 317.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z318.000318.000318.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-5.2018.0770.001

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Supplemental data

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Sample components

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Entire : ClpXP complex

EntireName: ClpXP complex
Components
  • Complex: ClpXP complex

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Supramolecule #1: ClpXP complex

SupramoleculeName: ClpXP complex / type: complex / ID: 1 / Parent: 0
Details: Complex formed between ClpX hexmers and a ClpP tetradecamer
Source (natural)Organism: Neisseria meningitidis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Bl21(DE3) / Recombinant plasmid: pet28a
Molecular weightTheoretical: 860 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

Concentration2 mg/mL
BufferpH: 8.5
Component:
ConcentrationName
50.0 mMBicine
100.0 mMPotassium Chloride
2.0 mMATPAdenosine triphosphate
2.0 mMMagnesium Chloride

Details: Buffer pH was measured as 8.2 at room temperature corresponding to a pH of 8.5 at 4 degrees, the temperature at which the complex was held before vitrification
GridSupport film - topology: HOLEY / Support film - Film thickness: 30.0 nm / Details: unspecified
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III / Details: Blotted for 15 seconds at an offset of -5 mm.
DetailsMono-disperse complexes

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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: 2680 / Average exposure time: 60.0 sec. / Average electron dose: 43.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 466549
CTF correctionSoftware - Name: cryoSPARC (ver. 2.10)
Startup modelType of model: OTHER / Details: ab initio model in cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.10)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 2.10)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 2.10)
Final reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.10) / Number images used: 377234

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Atomic model buiding 1

Initial modelPDB ID:

3hws

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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