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- EMDB-14443: E. coli C-P lyase bound to a PhnK ABC dimer in an open conformation -

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Entry
Database: EMDB / ID: EMD-14443
TitleE. coli C-P lyase bound to a PhnK ABC dimer in an open conformation
Map data
Sample
  • Complex: E. coli C-P lyase bound to a PhnK ABC dimer in an open conformation
    • Protein or peptide: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnG
    • Protein or peptide: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnH
    • Protein or peptide: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnI
    • Protein or peptide: Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P lyase
    • Protein or peptide: Putative phosphonates utilization ATP-binding protein PhnK
  • Ligand: ZINC ION
  • Ligand: alpha-D-ribose-1,2-cyclic-phosphate-5-phosphate
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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.57 Å
AuthorsAmstrup SK / Sofos N / Karlsen JL / Skjerning RB / Boesen T / Enghild JJ / Hove-Jensen B / Brodersen DE
Funding support Denmark, 1 items
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 SK / Sofos N / Karlsen JL / Skjerning RB / Boesen T / Enghild JJ / Hove-Jensen B / Brodersen DE
History
DepositionFeb 24, 2022-
Header (metadata) releaseMay 25, 2022-
Map releaseMay 25, 2022-
UpdateMar 8, 2023-
Current statusMar 8, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14443.png

Downloads & links

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Map

FileDownload / File: emd_14443.map.gz / Format: CCP4 / Size: 219.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.92 Å/pix.
x 386 pix.
= 355.85 Å		0.92 Å/pix.
x 386 pix.
= 355.85 Å		0.92 Å/pix.
x 386 pix.
= 355.85 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.92189 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.135
Minimum - Maximum-0.5260884 - 1.056952
Average (Standard dev.)0.00052055495 (±0.02264289)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions386386386
Spacing386386386
CellA=B=C: 355.85 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Map filtered to local resolution

Fileemd_14443_additional_1.map
AnnotationMap filtered to local resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_14443_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_14443_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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

EntireName: E. coli C-P lyase bound to a PhnK ABC dimer in an open conformation
Components
  • Complex: E. coli C-P lyase bound to a PhnK ABC dimer in an open conformation
    • Protein or peptide: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnG
    • Protein or peptide: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnH
    • Protein or peptide: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnI
    • Protein or peptide: Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P lyase
    • Protein or peptide: Putative phosphonates utilization ATP-binding protein PhnK
  • Ligand: ZINC ION
  • Ligand: alpha-D-ribose-1,2-cyclic-phosphate-5-phosphate

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Supramolecule #1: E. coli C-P lyase bound to a PhnK ABC dimer in an open conformation

SupramoleculeName: E. coli C-P lyase bound to a PhnK ABC dimer in an open conformation
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#5
Details: Post ATP hydrolysis by the ATP binding cassette (ABC) protein PhnK
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: Cytoplasm
Molecular weightTheoretical: 277 KDa

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Macromolecule #1: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subuni...

MacromoleculeName: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnG
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 16.545666 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MHADTATRQH WMSVLAHSQP AELAARLNAL NITADYEVIR AAETGLVQIQ ARMGGTGERF FAGDATLTRA AVRLTDGTLG YSWVLGRDK QHAERCALID ALMQQSRHFQ NLSETLIAPL DADRMARIAA RQAEVNASRV DFFTMVRGDN A

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Macromolecule #2: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subuni...

MacromoleculeName: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnH
type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
EC number: alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 21.045207 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTLETAFMLP VQDAQHSFRR LLKAMSEPGV IVALHQLKRG WQPLNIATTS VLLTLADNDT PVWLSTPLNN DIVNQSLRFH TNAPLVSQP EQATFAVTDE AISSEQLNAL STGTAVAPEA GATLILQVAS LSGGRMLRLT GAGIAEERMI APQLPECILH E LTERPHPF ...String:
MTLETAFMLP VQDAQHSFRR LLKAMSEPGV IVALHQLKRG WQPLNIATTS VLLTLADNDT PVWLSTPLNN DIVNQSLRFH TNAPLVSQP EQATFAVTDE AISSEQLNAL STGTAVAPEA GATLILQVAS LSGGRMLRLT GAGIAEERMI APQLPECILH E LTERPHPF PLGIDLILTC GERLLAIPRT THVEVC

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Macromolecule #3: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subuni...

MacromoleculeName: Alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase subunit PhnI
type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
EC number: alpha-D-ribose 1-methylphosphonate 5-triphosphate synthase
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 38.953742 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MYVAVKGGEK AIDAAHALQE SRRRGDTDLP ELSVAQIEQQ LNLAVDRVMT EGGIADRELA ALALKQASGD NVEAIFLLRA YRTTLAKLA VSEPLDTTGM RLERRISAVY KDIPGGQLLG PTYDYTHRLL DFTLLANGEA PTLTTADSEQ QPSPHVFSLL A RQGLAKFE ...String:
MYVAVKGGEK AIDAAHALQE SRRRGDTDLP ELSVAQIEQQ LNLAVDRVMT EGGIADRELA ALALKQASGD NVEAIFLLRA YRTTLAKLA VSEPLDTTGM RLERRISAVY KDIPGGQLLG PTYDYTHRLL DFTLLANGEA PTLTTADSEQ QPSPHVFSLL A RQGLAKFE EDSGAQPDDI TRTPPVYPCS RSSRLQQLMR GDEGYLLALA YSTQRGYGRN HPFAGEIRSG YIDVSIVPEE LG FAVNVGE LLMTECEMVN GFIDPPDEPP HFTRGYGLVF GMSERKAMAM ALVDRALQAP EYGEHATGPA QDEEFVLAHA DNV EAAGFV SHLKLPHYVD FQAELELLKR LQQEKNHG

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Macromolecule #4: Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P lyase

MacromoleculeName: Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P lyase
type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
EC number: alpha-D-ribose 1-methylphosphonate 5-phosphate C-P-lyase
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 31.893115 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MANLSGYNFA YLDEQTKRMI RRAILKAVAI PGYQVPFGGR EMPMPYGWGT GGIQLTASVI GESDVLKVID QGADDTTNAV SIRNFFKRV TGVNTTERTD DATLIQTRHR IPETPLTEDQ IIIFQVPIPE PLRFIEPRET ETRTMHALEE YGVMQVKLYE D IARFGHIA ...String:
MANLSGYNFA YLDEQTKRMI RRAILKAVAI PGYQVPFGGR EMPMPYGWGT GGIQLTASVI GESDVLKVID QGADDTTNAV SIRNFFKRV TGVNTTERTD DATLIQTRHR IPETPLTEDQ IIIFQVPIPE PLRFIEPRET ETRTMHALEE YGVMQVKLYE D IARFGHIA TTYAYPVKVN GRYVMDPSPI PKFDNPKMDM MPALQLFGAG REKRIYAVPP FTRVESLDFD DHPFTVQQWD EP CAICGST HSYLDEVVLD DAGNRMFVCS DTDYCRQQSE AKNQ

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Macromolecule #5: Putative phosphonates utilization ATP-binding protein PhnK

MacromoleculeName: Putative phosphonates utilization ATP-binding protein PhnK
type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 31.995146 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNQPLLSVNN LTHLYAPGKG FSDVSFDLWP GEVLGIVGES GSGKTTLLKS ISARLTPQQG EIHYENRSLY AMSEADRRRL LRTEWGVVH QHPLDGLRRQ VSAGGNIGER LMATGARHYG DIRATAQKWL EEVEIPANRI DDLPTTFSGG MQQRLQIARN L VTHPKLVF ...String:
MNQPLLSVNN LTHLYAPGKG FSDVSFDLWP GEVLGIVGES GSGKTTLLKS ISARLTPQQG EIHYENRSLY AMSEADRRRL LRTEWGVVH QHPLDGLRRQ VSAGGNIGER LMATGARHYG DIRATAQKWL EEVEIPANRI DDLPTTFSGG MQQRLQIARN L VTHPKLVF MDEPTGGLDV SVQARLLDLL RGLVVELNLA VVIVTHDLGV ARLLADRLLV MKQGQVVESG LTDRVLDDPH HP YTQLLVS SVLQNENLYF QGQFGSWSHP QFEKGGGSGG GSGGGSWSHP QFEK

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #7: alpha-D-ribose-1,2-cyclic-phosphate-5-phosphate

MacromoleculeName: alpha-D-ribose-1,2-cyclic-phosphate-5-phosphate / type: ligand / ID: 7 / Number of copies: 1 / Formula: I9X
Molecular weightTheoretical: 292.074 Da
Chemical component information

ChemComp-I8Z:
Alpha-D-ribose-1,2-cyclic-phosphate-5-phosphate

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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

Concentration2.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPES/KOH(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
125.0 mMKClpotassium chloride
0.5 mMEDTAEthylenediaminetetraacetic acidEthylenediaminetetraacetic acid
5.0 mMBMEbeta mercaptoethanol
1.5 mMATPAdenosine triphosphateAdenine triphosphate
6.0 mMMgCl2Magnesium chloride

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
GridModel: UltrAuFoil R0.6/1 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR / Details: 10 mA in an GloQube, Quorum
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 310 K / Instrument: LEICA EM GP / Details: Blotting for 6-9 seconds before plunging.
DetailsSample was produced under ATP to ADP+Pi turnover conditions. Sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.4000000000000001 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 135000
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 62.0 e/Å2
Details: Images were collected in movie-mode with a total of 56 frames
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1155385 / Details: Deep picker from cryoSPARC used
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2) / Software - details: Ab-initio
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2) / Software - details: Non-uniform refinement
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.57 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 31280
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4xb6

RefinementSpace: RECIPROCAL / Overall B value: 28.7
Output model

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

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