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- EMDB-3033: Structure of PhnGHIJK complex by negative stain electron microscopy -

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Basic information

Entry
Database: EMDB / ID: EMD-3033
TitleStructure of PhnGHIJK complex by negative stain electron microscopy
Map data3D reconstruction of C-P lyase core complex including K subunit
Sample
  • Sample: Escherichia coli PhnGHIJK complex
  • Protein or peptide: PhnG
  • Protein or peptide: PhnH
  • Protein or peptide: PhnI
  • Protein or peptide: PhnJ
  • Protein or peptide: PhnK
Keywordsphosphorus metabolism / carbon-phosphorous lyase / phosphonate / PhnG / PhnH / PhnI / PhnJ / PhnK
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 16.0 Å
AuthorsSeweryn P / Bich Van L / Kjeldgaard M / Russo CJ / Passmore LA / Hove-Jensen B / Jochimsen B / Brodersen DE
CitationJournal: Nature / Year: 2015
Title: Structural insights into the bacterial carbon-phosphorus lyase machinery.
Authors: Paulina Seweryn / Lan Bich Van / Morten Kjeldgaard / Christopher J Russo / Lori A Passmore / Bjarne Hove-Jensen / Bjarne Jochimsen / Ditlev E Brodersen /
Abstract: Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use ...Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C-P lyase core complex (PhnG-PhnH-PhnI-PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that probably couple phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy, and show that it binds to a conserved insertion domain of PhnJ. Our results provide a structural basis for understanding microbial phosphonate breakdown.
History
DepositionMay 30, 2015-
Header (metadata) releaseJul 15, 2015-
Map releaseAug 26, 2015-
UpdateSep 23, 2015-
Current statusSep 23, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.66
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.66
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3033.png

Downloads & links

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Map

FileDownload / File: emd_3033.map.gz / Format: CCP4 / Size: 670.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of C-P lyase core complex including K subunit
Voxel sizeX=Y=Z: 3.07 Å
Density
Contour LevelBy AUTHOR: 0.71 / Movie #1: 0.66
Minimum - Maximum-0.00770088 - 1.36574364
Average (Standard dev.)0.09359466 (±0.21601628)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions565656
Spacing565656
CellA=B=C: 171.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.073.073.07
M x/y/z565656
origin x/y/z0.0000.0000.000
length x/y/z171.920171.920171.920
α/β/γ90.00090.00090.000
start NX/NY/NZ-21-120
NX/NY/NZ432573
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS565656
D min/max/mean-0.0081.3660.094

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Supplemental data

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Sample components

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Entire : Escherichia coli PhnGHIJK complex

EntireName: Escherichia coli PhnGHIJK complex
Components
  • Sample: Escherichia coli PhnGHIJK complex
  • Protein or peptide: PhnG
  • Protein or peptide: PhnH
  • Protein or peptide: PhnI
  • Protein or peptide: PhnJ
  • Protein or peptide: PhnK

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Supramolecule #1000: Escherichia coli PhnGHIJK complex

SupramoleculeName: Escherichia coli PhnGHIJK complex / type: sample / ID: 1000 / Number unique components: 5
Molecular weightTheoretical: 268 KDa

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Macromolecule #1: PhnG

MacromoleculeName: PhnG / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: heterodimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 21 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pHO575

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Macromolecule #2: PhnH

MacromoleculeName: PhnH / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Oligomeric state: heterodimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 21 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pHO575

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Macromolecule #3: PhnI

MacromoleculeName: PhnI / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Oligomeric state: heterodimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 39 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pHO575

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Macromolecule #4: PhnJ

MacromoleculeName: PhnJ / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Oligomeric state: heterodimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 32 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pHO575

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Macromolecule #5: PhnK

MacromoleculeName: PhnK / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 28 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pHO575

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

StainingType: NEGATIVE
Details: 3% ammonium molybdate pH 8.0 followed by 2% uranyl acetate
GridDetails: Quantifoil R2/2 holey carbon on copper mesh grids, covered with an additional thin film of amorphous carbon
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.974 µm / Nominal defocus min: 0.832 µm / Nominal magnification: 44000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateJul 26, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 105 / Average electron dose: 20 e/Å2

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Image processing #1

CTF correctionDetails: CTFFIND3
Final reconstructionResolution.type: BY AUTHOR / Resolution: 28.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Relion / Number images used: 10033
DetailsParticles manually picked using EMAN Boxer. 2D and 3D processing in Relion.
Image processing ID1
FSC plot (resolution estimation)

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Image processing #2

CTF correctionDetails: CTFFIND3
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 10033
DetailsParticles manually picked using EMAN Boxer. 2D and 3D processing in Relion.
Image processing ID2
FSC plot (resolution estimation)

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