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- EMDB-5903: 3D Reconstruction of Membrane Protein Complex ExbB4-ExbD2 -

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Basic information

Entry
Database: EMDB / ID: 5903
Title3D Reconstruction of Membrane Protein Complex ExbB4-ExbD2
Map dataExbB4-ExbD2 complex with ExbD periplasmic domains dimerized in the membrane-parallel position
SampleMembrane protein complex ExbB4-ExbD2 from Escherichia coliBiological membrane:
(Biopolymer Transport Protein ...) x 2
KeywordsMembrane protein complex / coordinated rearrangement / flexible domain
Function / homologyMotA/TolQ/ExbB proton channel family / Biopolymer transport protein ExbD/TolR / TonB system transport protein ExbD type-1 / TonB-system energizer ExbB type-1 / Biopolymer transport protein ExbD/TolR / MotA/TolQ/ExbB proton channel / TonB system transport protein ExbD type-1 / TonB-system energizer ExbB type-1 / energy transducer activity / bacteriocin transport ...MotA/TolQ/ExbB proton channel family / Biopolymer transport protein ExbD/TolR / TonB system transport protein ExbD type-1 / TonB-system energizer ExbB type-1 / Biopolymer transport protein ExbD/TolR / MotA/TolQ/ExbB proton channel / TonB system transport protein ExbD type-1 / TonB-system energizer ExbB type-1 / energy transducer activity / bacteriocin transport / plasma membrane protein complex / transmembrane transporter activity / protein transport / protein stabilization / integral component of plasma membrane / integral component of membrane / identical protein binding / plasma membrane / Biopolymer transport protein ExbB / Biopolymer transport protein ExbD
Function and homology information
SourceEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / 24 Å resolution
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.
DateDeposition: Feb 12, 2014 / Header (metadata) release: Mar 5, 2014 / Map release: Apr 9, 2014 / Last update: Feb 17, 2016

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Structure visualization

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

Downloads & links

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Map

Fileemd_5903.map.gz (map file in CCP4 format, 8193 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
128 pix
2.2 Å/pix.
= 281.6 Å
128 pix
2.2 Å/pix.
= 281.6 Å
128 pix
2.2 Å/pix.
= 281.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2 Å
Density
Contour Level:0.036 (by author), 0.036 (movie #1):
Minimum - Maximum-0.08121978 - 0.09978295
Average (Standard dev.)-0.00111288 (0.01272697)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions128128128
Origin000
Limit127127127
Spacing128128128
CellA=B=C: 281.6 Å
α=β=γ: 90 deg.

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
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0810.100-0.001

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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 coli
Number of components: 2 / Oligomeric State: Four ExbB in complex with two ExbD
MassTheoretical: 139 kDa

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Component #1: protein, Biopolymer Transport Protein ExbB

ProteinName: Biopolymer Transport Protein ExbB / a.k.a: ExbB / Oligomeric Details: Tetramer / Number of Copies: 4 / Recombinant expression: Yes
MassTheoretical: 26 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: K-12
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pQE60 / Strain: M15
Source (natural)Location in cell: Cytoplasmic Membrane
External referencesGene Ontology: integral component of membrane / UniProt: Biopolymer transport protein ExbB / InterPro: TonB-system energizer ExbB type-1

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Component #2: protein, Biopolymer Transport Protein ExbD

ProteinName: Biopolymer Transport Protein ExbD / a.k.a: ExbD / Oligomeric Details: Dimer / Recombinant expression: Yes / Number of Copies: 2
MassTheoretical: 17 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: K-12
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pQE60 / Strain: M15
Source (natural)Location in cell: Cytoplasmic Membrane
External referencesInterPro: TonB system transport protein ExbD type-1 / UniProt: Biopolymer transport protein ExbD / Gene Ontology: integral component of membrane

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: negative staining
Sample solutionSpecimen conc.: 0.02 mg/ml / Buffer solution: 25 mM Tris-HCl, 150 mM NaCl, 0.01% DDM / pH: 7.5
Support film400 mesh copper grid with thin carbon support, glow discharged
StainingGrids with adsorbed protein were stained with 1.5% uranyl formate for 1 minute.
VitrificationInstrument: NONE / Cryogen name: NONE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Mar 27, 2013
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 67000 X (nominal), 67147 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 50,000x magnification
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 3500 nm
Specimen HolderHolder: Room temperature / Model: SIDE ENTRY, EUCENTRIC
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 5562
Details: Manual particle picking from random conical tilt pairs, 3D classification, projection matching angular refinement
3D reconstructionAlgorithm: Random conical tilt, ML3D classification / Software: Xmipp, EMAN2, SPARX / CTF correction: Each Micrograph / Resolution: 24 Å / Resolution method: FSC 0.5, semi-independent

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