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- PDB-7z17: E. coli C-P lyase bound to a PhnK ABC dimer in an open conformation -

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Basic information

Entry
Database: PDB / ID: 7z17
TitleE. coli C-P lyase bound to a PhnK ABC dimer in an open conformation
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 / ATP hydrolysis activity / protein homodimerization activity / ATP binding / identical protein binding / metal ion binding
Similarity search - Function
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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
alpha-D-ribose-1,2-cyclic-phosphate-5-phosphate / 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 2.0Jun 22, 2022Group: Atomic model / Author supporting evidence ...Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / audit_author ...atom_site / audit_author / chem_comp / citation / citation_author / entity / pdbx_entity_instance_feature / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_conn_angle / struct_conn
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.pdbx_formal_charge / _audit_author.identifier_ORCID / _chem_comp.id / _chem_comp.name / _chem_comp.pdbx_synonyms / _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.identifier_ORCID / _citation_author.name / _entity.pdbx_description / _pdbx_entity_instance_feature.auth_comp_id / _pdbx_entity_instance_feature.comp_id / _pdbx_entity_nonpoly.comp_id / _pdbx_entity_nonpoly.name / _pdbx_nonpoly_scheme.mon_id / _pdbx_nonpoly_scheme.pdb_mon_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id
Revision 2.1Mar 8, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model

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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
J: Putative phosphonates utilization ATP-binding protein PhnK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)281,41915
Polymers280,86610
Non-polymers5545
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Negative stain
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: 16545.666 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: pET28(a) / Production host: Escherichia coli (E. coli) / Strain (production host): Lemo21
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: 21045.207 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: pET28(a) / Production host: Escherichia coli (E. coli) / Strain (production host): Lemo21
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: 38953.742 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: phnI, b4099, JW4060 / Plasmid: pET28(a) / Production host: Escherichia coli (E. coli) / Strain (production host): Lemo21
References: UniProt: P16687, alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase

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Protein , 2 types, 4 molecules DHIJ

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


Mass: 31893.115 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: phnJ, b4098, JW4059 / Plasmid: pET28(a) / Production host: Escherichia coli (E. coli) / Strain (production host): Lemo21
References: UniProt: P16688, alpha-D-ribose 1-methylphosphonate 5-phosphate C-P-lyase
#5: Protein Putative phosphonates utilization ATP-binding protein PhnK


Mass: 31995.146 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: phnK, b4097, JW5727 / Plasmid: pET28(a) / Production host: Escherichia coli (E. coli) / Strain (production host): Lemo21 / References: UniProt: P16678

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Non-polymers , 2 types, 5 molecules

#6: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-I9X / alpha-D-ribose-1,2-cyclic-phosphate-5-phosphate / [(3aR,5R,6R,6aR)-2,6-bis(oxidanyl)-2-oxidanylidene-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3,2]dioxaphosphol-5-yl]methyl dihydrogen phosphate / 5-phospho-alpha-D-ribose cyclic-1,2-phosphate


Mass: 292.074 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H10O10P2 / Feature type: SUBJECT OF INVESTIGATION

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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

ComponentName: E. coli C-P lyase bound to a PhnK ABC dimer in an open conformation
Type: COMPLEX
Details: Post ATP hydrolysis by the ATP binding cassette (ABC) protein PhnK
Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.277 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli) / Cellular location: Cytoplasm
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Lemo21 / Plasmid: pET28(a)
Buffer solutionpH: 7.5
Details: ATP up from a concentration from 0.25 to 1.5 and MgCl2 to a concentration of 6 mM was added 15 seconds before the sample was plunge frozen
Buffer component
IDConc.NameFormulaBuffer-ID
125 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES/KOH1
2125 mMpotassium chlorideKCl1
30.5 mMEthylenediaminetetraacetic acidEDTAEthylenediaminetetraacetic acid1
45 mMbeta mercaptoethanolBME1
51.5 mMAdenine triphosphateATPAdenosine triphosphate1
66 mMMagnesium chlorideMgCl21
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was produced under ATP to ADP+Pi turnover conditions. Sample was monodisperse.
Specimen supportDetails: 10 mA in an GloQube, Quorum / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R0.6/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 310 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 FIELDBright-field microscopy / Nominal magnification: 135000 X / Nominal defocus max: 1400 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Image recordingElectron dose: 62 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1
Details: Images were collected in movie-mode with a total of 56 frames
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4cryoSPARC3.2CTF correction
9cryoSPARC3.2initial Euler assignmentAb-initio
10cryoSPARC3.2final Euler assignmentNon-uniform refinement
11cryoSPARC3.2classification3D variability
12cryoSPARC3.23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1155385 / Details: Deep picker from cryoSPARC used
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.57 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31280 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 28.7 / Space: RECIPROCAL
Atomic model buildingPDB-ID: 4XB6

4xb6

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