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- EMDB-6412: Structure of PhnGI complex from Escherichia coli by negative stain -

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Basic information

Entry
Database: EMDB / ID: EMD-6412
TitleStructure of PhnGI complex from Escherichia coli by negative stain
Map dataPhnGI complex from Escherichia coli
Sample
  • Sample: PhnGI complex from Escherichia coli
  • Protein or peptide: PhnG
  • Protein or peptide: PhnI
KeywordsPhnGI
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsYang K / Ren Z / Raushel FM / Zhang J
CitationJournal: Structure / Year: 2016
Title: Structures of the Carbon-Phosphorus Lyase Complex Reveal the Binding Mode of the NBD-like PhnK.
Authors: Kailu Yang / Zhongjie Ren / Frank M Raushel / Junjie Zhang /
Abstract: The carbon-phosphorus (C-P) lyase complex is essential for the metabolism of unactivated phosphonates to phosphate in bacteria. Using single-particle cryo-electron microscopy, we determined two ...The carbon-phosphorus (C-P) lyase complex is essential for the metabolism of unactivated phosphonates to phosphate in bacteria. Using single-particle cryo-electron microscopy, we determined two structures of the C-P lyase core complex PhnG2H2I2J2, with or without PhnK. PhnG2H2I2J2 is a two-fold symmetric hetero-octamer. Its two PhnJ subunits provide two identical binding sites for PhnK. Only one PhnK binds to PhnG2H2I2J2 due to steric hindrance. PhnK is homologous to the nucleotide-binding domain (NBD) of ATP-binding cassette transporters. The α helices 3 and 4 of PhnK bind to α helix 6 and a loop (residues 227-230) of PhnJ, in a different mode from the binding of NBDs to their transmembrane partners. Moreover, binding of PhnK exposes the active site residue, Gly32 of PhnJ, located near the interface between PhnJ and PhnH. This structural information provides a basis for further deciphering of the reaction mechanism of the C-P lyase.
History
DepositionAug 2, 2015-
Header (metadata) releaseAug 12, 2015-
Map releaseAug 12, 2015-
UpdateAug 12, 2015-
Current statusAug 12, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6412.png

Downloads & links

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Map

FileDownload / File: emd_6412.map.gz / Format: CCP4 / Size: 104.5 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPhnGI complex from Escherichia coli
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.3 Å/pix.
x 30 pix.
= 159. Å		5.3 Å/pix.
x 30 pix.
= 159. Å		5.3 Å/pix.
x 30 pix.
= 159. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-0.03472968 - 0.94091636
Average (Standard dev.)0.06531542 (±0.17226292)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions303030
Spacing303030
CellA=B=C: 159.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.35.35.3
M x/y/z303030
origin x/y/z0.0000.0000.000
length x/y/z159.000159.000159.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS303030
D min/max/mean-0.0350.9410.065

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Supplemental data

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Sample components

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Entire : PhnGI complex from Escherichia coli

EntireName: PhnGI complex from Escherichia coli
Components
  • Sample: PhnGI complex from Escherichia coli
  • Protein or peptide: PhnG
  • Protein or peptide: PhnI

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Supramolecule #1000: PhnGI complex from Escherichia coli

SupramoleculeName: PhnGI complex from Escherichia coli / type: sample / ID: 1000 / Oligomeric state: Two PhnG, two PhnI / Number unique components: 2
Molecular weightTheoretical: 115 KDa

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Macromolecule #1: PhnG

MacromoleculeName: PhnG / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: BW5328
Molecular weightTheoretical: 18.7 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta2 (DE3) pLysS cells (Novagen) / Recombinant plasmid: pET-28b

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Macromolecule #2: PhnI

MacromoleculeName: PhnI / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: BW5328
Molecular weightTheoretical: 38.9 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta2 (DE3) pLysS cells (Novagen) / Recombinant plasmid: pET-28b

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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

Concentration0.01 mg/mL
BufferpH: 8.5 / Details: 50 mM HEPES, 150 mM NaCl, 2 mM TCEP
StainingType: NEGATIVE / Details: sample was stained by 2% uranyl acetate
GridDetails: 200 mesh copper grid with carbon film
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
DateMay 21, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Number real images: 20 / Average electron dose: 40 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 26285 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 4.1 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsParticles were selected in EMAN2. Classification and refinement were performed in Relion.
CTF correctionDetails: Each micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: CTFFIND4, EMAN2, Relion / Number images used: 2298
FSC plot (resolution estimation)

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