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- EMDB-21194: ClpXP from Neisseria meningitidis - Conformation B -

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Basic information

Entry
Database: EMDB / ID: EMD-21194
TitleClpXP from Neisseria meningitidis - Conformation B
Map dataClpXP from N. meningitidis - Conformation B
Sample
  • Complex: ClpXP complex
    • Complex: ATP-dependent Clp protease ATP-binding subunit ClpX, ATP-dependent Clp protease proteolytic subunit
      • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpX
      • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
    • Complex: Unidentified peptide substrate
      • Protein or peptide: Unidentified peptide substrate
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsComplex / AAA+ / protease / ClpP / ClpX / HYDROLASE
Function / homology
Function and homology information


HslUV protease complex / endopeptidase Clp complex / endopeptidase Clp / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / proteolysis involved in protein catabolic process / ATP-dependent protein folding chaperone / unfolded protein binding / peptidase activity / ATPase binding ...HslUV protease complex / endopeptidase Clp complex / endopeptidase Clp / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / proteolysis involved in protein catabolic process / ATP-dependent protein folding chaperone / unfolded protein binding / peptidase activity / ATPase binding / protein dimerization activity / cell division / serine-type endopeptidase activity / ATP hydrolysis activity / proteolysis / zinc ion binding / ATP binding / cytoplasm
Similarity search - Function
Clp protease, ATP-binding subunit ClpX, bacteria / Zinc finger, ClpX C4-type superfamily / ClpX C4-type zinc finger / Clp protease, ATP-binding subunit ClpX / Zinc finger, ClpX C4-type / ClpX zinc binding (ZB) domain profile. / ClpX C4-type zinc finger / ClpP, Ser active site / Endopeptidase Clp serine active site. / ClpP, histidine active site ...Clp protease, ATP-binding subunit ClpX, bacteria / Zinc finger, ClpX C4-type superfamily / ClpX C4-type zinc finger / Clp protease, ATP-binding subunit ClpX / Zinc finger, ClpX C4-type / ClpX zinc binding (ZB) domain profile. / ClpX C4-type zinc finger / 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 / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / ClpP/crotonase-like domain superfamily / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ATP-dependent Clp protease ATP-binding subunit ClpX / ATP-dependent Clp protease proteolytic subunit / ATP-dependent Clp protease ATP-binding subunit ClpX / ATP-dependent Clp protease proteolytic subunit
Similarity search - Component
Biological speciesNeisseria meningitidis (bacteria) / Escherichia coli (E. coli) / Escherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsRipstein ZA / Vahidi S
Funding support Canada, 3 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)FDN-503573 Canada
Canadian Institutes of Health Research (CIHR)PJT-162186 Canada
Canadian Institutes of Health Research (CIHR)PJT-148564 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-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vfx
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21194.png

Downloads & links

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Map

FileDownload / File: emd_21194.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClpXP from N. meningitidis - Conformation B
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.6 / Movie #1: 0.6
Minimum - Maximum-2.0846157 - 5.6985774
Average (Standard dev.)0.0012604895 (±0.11967329)
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-2.0855.6990.001

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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
    • Complex: ATP-dependent Clp protease ATP-binding subunit ClpX, ATP-dependent Clp protease proteolytic subunit
      • Protein or peptide: ATP-dependent Clp protease ATP-binding subunit ClpX
      • Protein or peptide: ATP-dependent Clp protease proteolytic subunit
    • Complex: Unidentified peptide substrate
      • Protein or peptide: Unidentified peptide substrate
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: ClpXP complex

SupramoleculeName: ClpXP complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Complex formed between ClpX hexmers and a ClpP tetradecamer
Molecular weightTheoretical: 860 KDa

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Supramolecule #2: ATP-dependent Clp protease ATP-binding subunit ClpX, ATP-dependen...

SupramoleculeName: ATP-dependent Clp protease ATP-binding subunit ClpX, ATP-dependent Clp protease proteolytic subunit
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #3
Source (natural)Organism: Neisseria meningitidis (bacteria)

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Supramolecule #3: Unidentified peptide substrate

SupramoleculeName: Unidentified peptide substrate / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia coli (E. coli) / Strain: Bl21(DE3)

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Macromolecule #1: ATP-dependent Clp protease ATP-binding subunit ClpX

MacromoleculeName: ATP-dependent Clp protease ATP-binding subunit ClpX / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Neisseria meningitidis (bacteria)
Molecular weightTheoretical: 45.139438 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSNENRTCSF CGKSKSHVKH LIEGENAFIC DECVSNCIEI LHEDGNDGTP SESAGGEPEE SGKLPTPAEI VANLNDHVIG QEQAKKALA VSVYNHYKRL RHPKAGANVE LSKSNILLIG PTGSGKTLLA QSLARKLDVP FVMADATTLT EAGYVGEDVE Q IITKLLGK ...String:
MSNENRTCSF CGKSKSHVKH LIEGENAFIC DECVSNCIEI LHEDGNDGTP SESAGGEPEE SGKLPTPAEI VANLNDHVIG QEQAKKALA VSVYNHYKRL RHPKAGANVE LSKSNILLIG PTGSGKTLLA QSLARKLDVP FVMADATTLT EAGYVGEDVE Q IITKLLGK CDFDVEKAQR GIVYIDQIDK ISRKSDNPSI TRDVSGEGVQ QALLKLIEGT VASVPPQGGR KHPNQEFINV DT TNILFIC GGAFAGLEKV IRQRTEKGGI GFGASVHSKD ENADITKLFG IVEPEDLIKF GLIPELIGRL PVIATLEILD EDA LINILT EPKNALVKQY QALFGMENVE LEFEEGALRS IARQAMERKT GARGLRSIVE RCLLDTMYRL PDLKGLKKVV VGKA VIEEG REPELVFES

UniProtKB: ATP-dependent Clp protease ATP-binding subunit ClpX

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Macromolecule #2: Unidentified peptide substrate

MacromoleculeName: Unidentified peptide substrate / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 613.749 Da
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #3: ATP-dependent Clp protease proteolytic subunit

MacromoleculeName: ATP-dependent Clp protease proteolytic subunit / type: protein_or_peptide / ID: 3 / Number of copies: 14 / Enantiomer: LEVO / EC number: endopeptidase Clp
Source (natural)Organism: Neisseria meningitidis (bacteria)
Molecular weightTheoretical: 22.729967 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSFDNYLVPT VIEQSGRGER AFDIYSRLLK ERIVFLVGPV TDESANLVVA QLLFLESENP DKDIFFYINS PGGSVTAGMS IYDTMNFIK PDVSTLCLGQ AASMGAFLLS AGEKGKRFAL PNSRIMIHQP LISGGLGGQA SDIEIHAREL LKIKEKLNRL M AKHCDRDL ...String:
MSFDNYLVPT VIEQSGRGER AFDIYSRLLK ERIVFLVGPV TDESANLVVA QLLFLESENP DKDIFFYINS PGGSVTAGMS IYDTMNFIK PDVSTLCLGQ AASMGAFLLS AGEKGKRFAL PNSRIMIHQP LISGGLGGQA SDIEIHAREL LKIKEKLNRL M AKHCDRDL ADLERDTDRD NFMSAEEAKE YGLIDQILEN RASLRL

UniProtKB: ATP-dependent Clp protease proteolytic subunit

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 5 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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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 / 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
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: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.10) / Number images used: 178448

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Atomic model buiding 1

Initial modelPDB ID:

3hws


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

PDB-6vfx:
ClpXP from Neisseria meningitidis - Conformation B

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