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- EMDB-5901: 3D Reconstruction of Membrane Protein Complex ExbB4-ExbD2 -

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Basic information

Entry
Database: EMDB / ID: EMD-5901
Title3D Reconstruction of Membrane Protein Complex ExbB4-ExbD2
Map dataExbB4-ExbD2 complex with ExbD periplasmic domains unobservable
Sample
  • Sample: Membrane protein complex ExbB4-ExbD2 from Escherichia coliBiological membrane
  • Protein or peptide: Biopolymer Transport Protein ExbB
  • Protein or peptide: Biopolymer Transport Protein ExbD
KeywordsMembrane protein complex / coordinated rearrangement / flexible domain
Function / homology
Function and homology information


energy transducer activity / bacteriocin transport / cobalamin transport / intracellular monoatomic cation homeostasis / protein import / plasma membrane protein complex / transmembrane transporter complex / transmembrane transporter activity / cell outer membrane / protein transport ...energy transducer activity / bacteriocin transport / cobalamin transport / intracellular monoatomic cation homeostasis / protein import / plasma membrane protein complex / transmembrane transporter complex / transmembrane transporter activity / cell outer membrane / protein transport / intracellular iron ion homeostasis / membrane => GO:0016020 / protein stabilization / protein homodimerization activity / membrane / identical protein binding / plasma membrane
Similarity search - Function
TonB-system energizer ExbB type-1 / TonB-system energizer ExbB type-1 / TonB system transport protein ExbD type-1 / TonB system transport protein ExbD type-1 / Biopolymer transport protein ExbD/TolR / Biopolymer transport protein ExbD/TolR / MotA/TolQ/ExbB proton channel / MotA/TolQ/ExbB proton channel family
Similarity search - Domain/homology
Biopolymer transport protein ExbB / Biopolymer transport protein ExbD
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 Å
AuthorsSverzhinsky A / Fabre L / Cottreau AL / Biot-Pelletier DMP / Khalil S / Bostina M / Rouiller I / Coulton JW
CitationJournal: Structure / Year: 2014
Title: Coordinated rearrangements between cytoplasmic and periplasmic domains of the membrane protein complex ExbB-ExbD of Escherichia coli.
Authors: Aleksandr Sverzhinsky / Lucien Fabre / Andrew L Cottreau / Damien M P Biot-Pelletier / Sofia Khalil / Mihnea Bostina / Isabelle Rouiller / James W Coulton /
Abstract: Gram-negative bacteria rely on the ExbB-ExbD-TonB system for the import of essential nutrients. Despite decades of research, the stoichiometry, subunit organization, and mechanism of action of the ...Gram-negative bacteria rely on the ExbB-ExbD-TonB system for the import of essential nutrients. Despite decades of research, the stoichiometry, subunit organization, and mechanism of action of the membrane proteins of the Ton system remain unclear. We copurified ExbB with ExbD as an ∼240 kDa protein-detergent complex, measured by light scattering and by native gels. Quantitative Coomassie staining revealed a stoichiometry of ExbB4-ExbD2. Negative stain electron microscopy and 2D analysis showed particles of ∼10 nm diameter in multiple structural states. Nanogold labeling identified the position of the ExbD periplasmic domain. Random conical tilt was used to reconstruct the particles in three structural states followed by sorting of the single particles and refinement of each state. The different states are interpreted by coordinated structural rearrangements between the cytoplasmic domain and the periplasmic domain, concordant with in vivo predictions.
History
DepositionFeb 12, 2014-
Header (metadata) releaseMar 5, 2014-
Map releaseApr 9, 2014-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.029
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.036
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5901.gif

Downloads & links

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Map

FileDownload / File: emd_5901.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationExbB4-ExbD2 complex with ExbD periplasmic domains unobservable
Voxel sizeX=Y=Z: 2.2 Å
Density
Contour LevelBy AUTHOR: 0.029 / Movie #1: 0.029
Minimum - Maximum-0.09642076 - 0.10619523
Average (Standard dev.)-0.00112127 (±0.01226369)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.22.22.2
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z281.600281.600281.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0960.106-0.001

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Supplemental data

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Sample components

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Entire : Membrane protein complex ExbB4-ExbD2 from Escherichia coli

EntireName: Membrane protein complex ExbB4-ExbD2 from Escherichia coliBiological membrane
Components
  • Sample: Membrane protein complex ExbB4-ExbD2 from Escherichia coliBiological membrane
  • Protein or peptide: Biopolymer Transport Protein ExbB
  • Protein or peptide: Biopolymer Transport Protein ExbD

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Supramolecule #1000: Membrane protein complex ExbB4-ExbD2 from Escherichia coli

SupramoleculeName: Membrane protein complex ExbB4-ExbD2 from Escherichia coli
type: sample / ID: 1000 / Oligomeric state: Four ExbB in complex with two ExbD / Number unique components: 2
Molecular weightTheoretical: 139 KDa

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Macromolecule #1: Biopolymer Transport Protein ExbB

MacromoleculeName: Biopolymer Transport Protein ExbB / type: protein_or_peptide / ID: 1 / Name.synonym: ExbB / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12 / Location in cell: Cytoplasmic Membrane
Molecular weightTheoretical: 26 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: M15 / Recombinant plasmid: pQE60
SequenceUniProtKB: Biopolymer transport protein ExbB / GO: membrane => GO:0016020 / InterPro: TonB-system energizer ExbB type-1

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Macromolecule #2: Biopolymer Transport Protein ExbD

MacromoleculeName: Biopolymer Transport Protein ExbD / type: protein_or_peptide / ID: 2 / Name.synonym: ExbD / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12 / Location in cell: Cytoplasmic Membrane
Molecular weightTheoretical: 17 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: M15 / Recombinant plasmid: pQE60
SequenceUniProtKB: Biopolymer transport protein ExbD / GO: membrane => GO:0016020 / InterPro: TonB system transport protein ExbD type-1

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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

Concentration0.02 mg/mL
BufferpH: 7.5 / Details: 25 mM Tris-HCl, 150 mM NaCl, 0.01% DDM
StainingType: NEGATIVE
Details: Grids with adsorbed protein were stained with 1.5% uranyl formate for 1 minute.
GridDetails: 400 mesh copper grid with thin carbon support, glow discharged
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 67147 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 67000
Sample stageSpecimen holder: Room temperature / Specimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 50,000x magnification
DateMar 27, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each Micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: OTHER / Software - Name: Xmipp, EMAN2, SPARX / Number images used: 7322
DetailsManual particle picking from random conical tilt pairs, 3D classification, projection matching angular refinement

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