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- PDB-7z19: E. coli C-P lyase bound to a single PhnK ABC domain -

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Basic information

Entry
Database: PDB / ID: 7z19
TitleE. coli C-P lyase bound to a single PhnK ABC domain
Components
  • (Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit ...) x 3
  • Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P lyase
  • Putative phosphonates utilization ATP-binding protein PhnK
KeywordsTRANSFERASE / protein complex / ABC / hydrolase / lyase / carbon phosphorus
Function / homology
Function and homology information


alpha-D-ribose 1-methylphosphonate 5-phosphate C-P-lyase / alpha-D-ribose 1-methylphosphonate 5-phosphate C-P-lyase activity / alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase / alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase activity / alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase complex / carbon phosphorus lyase complex / organic phosphonate metabolic process / organic phosphonate transport / organic phosphonate catabolic process / peptide transport ...alpha-D-ribose 1-methylphosphonate 5-phosphate C-P-lyase / alpha-D-ribose 1-methylphosphonate 5-phosphate C-P-lyase activity / alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase / alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase activity / alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase complex / carbon phosphorus lyase complex / organic phosphonate metabolic process / organic phosphonate transport / organic phosphonate catabolic process / peptide transport / 4 iron, 4 sulfur cluster binding / lyase activity / protein homodimerization activity / ATP hydrolysis activity / ATP binding / metal ion binding / identical protein binding
Similarity search - Function
Bacterial phosphonate metabolism protein PhnH / Phosphonate C-P lyase system, PhnK / Phosphonate metabolism protein PhnI / Phosphonate metabolism PhnG / Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P-lyase / Bacterial phosphonate metabolism protein (PhnI) / Phosphonate metabolism protein PhnJ / Phosphonate metabolism protein PhnG / Bacterial phosphonate metabolism, PhnH / PhnH-like superfamily ...Bacterial phosphonate metabolism protein PhnH / Phosphonate C-P lyase system, PhnK / Phosphonate metabolism protein PhnI / Phosphonate metabolism PhnG / Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P-lyase / Bacterial phosphonate metabolism protein (PhnI) / Phosphonate metabolism protein PhnJ / Phosphonate metabolism protein PhnG / Bacterial phosphonate metabolism, PhnH / PhnH-like superfamily / Bacterial phosphonate metabolism protein (PhnH) / Oligopeptide/dipeptide ABC transporter, C-terminal / Oligopeptide/dipeptide transporter, C-terminal region / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
PHOSPHATE ION / Putative phosphonates utilization ATP-binding protein PhnK / Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnG / Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnH / Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnI / Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P lyase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.57 Å
AuthorsAmstrup, S.K. / Sofos, N. / Karlsen, J.L. / Skjerning, R.B. / Boesen, T. / Enghild, J.J. / Hove-Jensen, B. / Brodersen, D.E.
Funding support Denmark, 1items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF18OC0030646 Denmark
Citation
Journal: Nat Commun / Year: 2023
Title: Structural remodelling of the carbon-phosphorus lyase machinery by a dual ABC ATPase.
Authors: Søren K Amstrup / Sui Ching Ong / Nicholas Sofos / Jesper L Karlsen / Ragnhild B Skjerning / Thomas Boesen / Jan J Enghild / Bjarne Hove-Jensen / Ditlev E Brodersen /
Abstract: In Escherichia coli, the 14-cistron phn operon encoding carbon-phosphorus lyase allows for utilisation of phosphorus from a wide range of stable phosphonate compounds containing a C-P bond. As part ...In Escherichia coli, the 14-cistron phn operon encoding carbon-phosphorus lyase allows for utilisation of phosphorus from a wide range of stable phosphonate compounds containing a C-P bond. As part of a complex, multi-step pathway, the PhnJ subunit was shown to cleave the C-P bond via a radical mechanism, however, the details of the reaction could not immediately be reconciled with the crystal structure of a 220 kDa PhnGHIJ C-P lyase core complex, leaving a significant gap in our understanding of phosphonate breakdown in bacteria. Here, we show using single-particle cryogenic electron microscopy that PhnJ mediates binding of a double dimer of the ATP-binding cassette proteins, PhnK and PhnL, to the core complex. ATP hydrolysis induces drastic structural remodelling leading to opening of the core complex and reconfiguration of a metal-binding and putative active site located at the interface between the PhnI and PhnJ subunits.
#1: Journal: Biorxiv / Year: 2022
Title: Structural remodelling of the carbon-phosphorus lyase machinery by a dual ABC ATPase
Authors: Amstrup, S.K. / Sofos, N. / Karlsen, J.L. / Skjerning, R.B. / Boesen, T. / Enghild, J.J. / Hove-Jensen, B. / Brodersen, D.E.
History
DepositionFeb 24, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 25, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 22, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_ASTM ..._citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Mar 8, 2023Group: Data collection / Database references / Refinement description
Category: citation / citation_author ...citation / citation_author / em_imaging / pdbx_initial_refinement_model
Item: _em_imaging.nominal_defocus_max / _em_imaging.nominal_defocus_min
Revision 1.3Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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 / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnG
B: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnH
C: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnI
D: Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P lyase
E: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnG
F: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnH
G: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnI
H: Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P lyase
I: Putative phosphonates utilization ATP-binding protein PhnK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)245,99115
Polymers245,5399
Non-polymers4526
Water8,593477
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, negative stain, gel filtration, Elution volume fits complex size
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit ... , 3 types, 6 molecules AEBFCG

#1: Protein Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnG / RPnTP synthase subunit PhnG


Mass: 16560.637 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: pBW120 / Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: phnG, b4101, JW4062 / Plasmid: pHO575 / Production host: Escherichia coli (E. coli) / Strain (production host): HO2735
References: UniProt: P16685, alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase
#2: Protein Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnH / RPnTP synthase subunit PhnH


Mass: 21074.271 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: pBW120 / Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: phnH, b4100, JW4061 / Plasmid: pHO575 / Production host: Escherichia coli (E. coli) / Strain (production host): HO2735
References: UniProt: P16686, alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase
#3: Protein Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnI / RPnTP synthase subunit PhnI / Ribose 1-methylphosphonate 5-triphosphate synthase nucleosidase subunit


Mass: 38922.707 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: phnI, b4099, JW4060 / Plasmid: pHO575 / Production host: Escherichia coli (E. coli) / Strain (production host): HO2735
References: UniProt: P16687, alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase

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Protein , 2 types, 3 molecules DHI

#4: Protein Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P lyase / PRPn C-P lyase


Mass: 31879.088 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: phnJ, b4098, JW4059 / Plasmid: pHO575 / Production host: Escherichia coli (E. coli) / Strain (production host): HO2735
References: UniProt: P16688, alpha-D-ribose 1-methylphosphonate 5-phosphate C-P-lyase
#5: Protein Putative phosphonates utilization ATP-binding protein PhnK


Mass: 28665.652 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: phnK, b4097, JW5727 / Plasmid: pHO575 / Production host: Escherichia coli (E. coli) / Strain (production host): HO2735 / References: UniProt: P16678

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Non-polymers , 3 types, 483 molecules

#6: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#7: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 477 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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

ComponentName: E. coli C-P lyase bound to a single PhnK ABC domain / Type: COMPLEX
Details: Complex of Phn(GHIJ)2K with a single PhnK subunit bound to the PhnGHIJ core complex
Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.252 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli) / Cellular location: Cytoplasm
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: HO2735 / Plasmid: pHO575
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES/NaOH1
2125 mMsodium chlorideNaCl1
35 mMbeta mercaptoethanolBME1
SpecimenConc.: 1.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was monodisperse.
Specimen supportDetails: 15 mA / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K / Details: Blotting for 6-9 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 135000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 17088
Details: Images were collected in movie-mode with 0.2 seconds per frame with a total of 40 frames
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3particle selection
2SerialEMimage acquisition
4RELION3.1CTF correction
9RELION3.1initial Euler assignment
10cryoSPARC3.1final Euler assignment
11cryoSPARC3.1classification3D variability
12cryoSPARC3.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 20000000 / Details: LoG picker from relion.py
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.57 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50323 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 54.2 / Space: RECIPROCAL
Atomic model buildingPDB-ID: 4XB6

4xb6


Accession code: 4XB6 / Source name: PDB / Type: experimental model

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