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- PDB-6vfs: ClpXP from Neisseria meningitidis - Conformation A -

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Basic information

Entry
Database: PDB / ID: 6vfs
TitleClpXP from Neisseria meningitidis - Conformation A
Components
  • (ATP-dependent Clp protease ...) x 2
  • Unidentified protein substrate
KeywordsHYDROLASE / Complex / AAA+ / protease / ClpP / ClpX
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 / Helicase, Ruva Protein; domain 3 - #60 / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / 2-enoyl-CoA Hydratase; Chain A, domain 1 / 2-enoyl-CoA Hydratase; Chain A, domain 1 / ClpP/crotonase-like domain superfamily / Helicase, Ruva Protein; domain 3 / ATPase, AAA-type, core / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Alpha-Beta Complex / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / 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 BL21 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsRipstein, Z.A. / Vahidi, S. / Houry, W.A. / Rubinstein, J.L. / Kay, L.E.
Funding support Canada, 3items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_label_asym_id

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
E: ATP-dependent Clp protease ATP-binding subunit ClpX
A: ATP-dependent Clp protease ATP-binding subunit ClpX
F: ATP-dependent Clp protease ATP-binding subunit ClpX
C: ATP-dependent Clp protease ATP-binding subunit ClpX
B: ATP-dependent Clp protease ATP-binding subunit ClpX
D: ATP-dependent Clp protease ATP-binding subunit ClpX
G: Unidentified protein substrate
L: ATP-dependent Clp protease proteolytic subunit
O: ATP-dependent Clp protease proteolytic subunit
P: ATP-dependent Clp protease proteolytic subunit
Q: ATP-dependent Clp protease proteolytic subunit
R: ATP-dependent Clp protease proteolytic subunit
S: ATP-dependent Clp protease proteolytic subunit
T: ATP-dependent Clp protease proteolytic subunit
U: ATP-dependent Clp protease proteolytic subunit
H: ATP-dependent Clp protease proteolytic subunit
I: ATP-dependent Clp protease proteolytic subunit
J: ATP-dependent Clp protease proteolytic subunit
K: ATP-dependent Clp protease proteolytic subunit
M: ATP-dependent Clp protease proteolytic subunit
N: ATP-dependent Clp protease proteolytic subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)592,65031
Polymers589,67021
Non-polymers2,98010
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area88010 Å2
ΔGint-423 kcal/mol
Surface area168600 Å2

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Components

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ATP-dependent Clp protease ... , 2 types, 20 molecules EAFCBDLOPQRSTUHIJKMN

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


Mass: 45139.438 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis (bacteria)
Gene: clpX, COH52_00090, COI09_01415, ERS514410_00003, NCTC8554_00172
Plasmid: pet28a / Details (production host): 6His tag with TEV site / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A0Y4ZJG4, UniProt: Q9JYY3*PLUS
#3: Protein
ATP-dependent Clp protease proteolytic subunit / / Endopeptidase Clp


Mass: 22729.967 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis (bacteria) / Gene: clpP, COI09_01760, ERS514410_00057 / Plasmid: pet28a / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: A0A0Y5K536, UniProt: Q9JZ38*PLUS, endopeptidase Clp

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Protein/peptide , 1 types, 1 molecules G

#2: Protein/peptide Unidentified protein substrate


Mass: 613.749 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)

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Non-polymers , 3 types, 10 molecules

#4: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1ClpXP complexCOMPLEXComplex formed between ClpX hexmers and a ClpP tetradecamer#1-#30RECOMBINANT
2ATP-dependent Clp protease ATP-binding subunit ClpX, ATP-dependent Clp protease proteolytic subunitCOMPLEX#1, #31RECOMBINANT
3Un-identified protein substrateCOMPLEX#21NATURAL
Molecular weightValue: 0.86 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Neisseria meningitidis (bacteria)487
23Escherichia coli (E. coli)469008Bl21(DE3)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Bl21(DE3) / Plasmid: pet28a
Buffer solutionpH: 8.5
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
Buffer component
IDConc.NameBuffer-ID
150 mMBicine1
2100 mMPotassium Chloride1
32 mMATPAdenosine triphosphate1
42 mMMagnesium Chloride1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Mono-disperse complexes
Specimen supportDetails: Home-made gold grids were inspected before use / Grid material: COPPER/RHODIUM / Grid mesh size: 400 divisions/in. / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blotted for 15 seconds at an offset of -5 mm

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 1700 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 70 K
Image recordingAverage exposure time: 60 sec. / Electron dose: 43 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2680
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.1particle selection
2EPUimage acquisition
4cryoSPARC2.1CTF correction
7UCSF Chimeramodel fitting
9cryoSPARC2.1initial Euler assignment
10cryoSPARC2.1final Euler assignment
11cryoSPARC2.1classification
12cryoSPARC2.13D reconstruction
13RosettaEMmodel refinement
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 466549
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110696 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 3HWS

3hws


Accession code: 3HWS / Source name: PDB / Type: experimental model

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