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- EMDB-2935: 3D Reconstruction of Membrane Protein Complex ExbB4-ExbD1-TonB1 -

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Basic information

Entry
Database: EMDB / ID: EMD-2935
Title3D Reconstruction of Membrane Protein Complex ExbB4-ExbD1-TonB1
Map dataExbB4-ExbD1-TonB1 showing cytoplasmic tetrameric arrangement and no periplasmic dimerization
Sample
  • Sample: Membrane protein complex ExbB4-ExbD1-TonB1 from Escherichia coliBiological membrane
  • Protein or peptide: Biopolymer Transport Protein ExbB
  • Protein or peptide: Biopolymer Transport Protein ExbD
  • Protein or peptide: Protein TonB
KeywordsMembrane protein complex / heterodimerization / coordinated rearrangement / iron import
Function / homology
Function and homology information


receptor-mediated bacteriophage irreversible attachment to host cell / colicin transport / energy transducer activity / cell envelope / bacteriocin transport / cobalamin transport / siderophore transport / intracellular monoatomic cation homeostasis / protein import / plasma membrane protein complex ...receptor-mediated bacteriophage irreversible attachment to host cell / colicin transport / energy transducer activity / cell envelope / bacteriocin transport / cobalamin transport / siderophore transport / intracellular monoatomic cation homeostasis / protein import / plasma membrane protein complex / transmembrane transporter complex / transmembrane transporter activity / cell outer membrane / transmembrane transport / protein transport / outer membrane-bounded periplasmic space / intracellular iron ion homeostasis / membrane => GO:0016020 / protein stabilization / protein domain specific binding / protein homodimerization activity / membrane / identical protein binding / plasma membrane
Similarity search - Function
TonB polyproline region / TonB C-terminal domain profile. / Gram-negative bacterial TonB protein / TonB, C-terminal / Gram-negative bacterial TonB protein C-terminal / TonB-system energizer ExbB type-1 / TonB-system energizer ExbB type-1 / TonB/TolA, C-terminal / TonB system transport protein ExbD type-1 / TonB/TolA, C-terminal ...TonB polyproline region / TonB C-terminal domain profile. / Gram-negative bacterial TonB protein / TonB, C-terminal / Gram-negative bacterial TonB protein C-terminal / TonB-system energizer ExbB type-1 / TonB-system energizer ExbB type-1 / TonB/TolA, C-terminal / TonB system transport protein ExbD type-1 / TonB/TolA, C-terminal / 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
Protein TonB / Biopolymer transport protein ExbB / Biopolymer transport protein ExbD
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsSverzhinsky A / Chung JWC / Deme JC / Fabre L / Levey KT / Plesa M / Carter DM / Lypaczewski P / Coulton JW
CitationJournal: J Bacteriol / Year: 2015
Title: Membrane Protein Complex ExbB4-ExbD1-TonB1 from Escherichia coli Demonstrates Conformational Plasticity.
Authors: Aleksandr Sverzhinsky / Jacqueline W Chung / Justin C Deme / Lucien Fabre / Kristian T Levey / Maria Plesa / David M Carter / Patrick Lypaczewski / James W Coulton /
Abstract: Iron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, ...Iron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His6-tagged exbB-exbD and S-tagged tonB, we purified detergent-solubilized complexes of ExbB-ExbD-TonB from Escherichia coli. Protein-detergent complexes of ∼230 kDa with a hydrodynamic radius of ∼6.0 nm were similar to previously purified ExbB₄-ExbD₂ complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB₄-ExbD₁-TonB₁. Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB₄-ExbD₁-TonB₁. Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization.
IMPORTANCE: Receptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and ...IMPORTANCE: Receptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstrate in vitro evidence of ExbB₄-ExbD₁-TonB₁ complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.
History
DepositionMar 13, 2015-
Header (metadata) releaseApr 1, 2015-
Map releaseApr 8, 2015-
UpdateMay 20, 2015-
Current statusMay 20, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.037
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.037
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2935.tif

Downloads & links

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Map

FileDownload / File: emd_2935.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationExbB4-ExbD1-TonB1 showing cytoplasmic tetrameric arrangement and no periplasmic dimerization
Voxel sizeX=Y=Z: 2.2 Å
Density
Contour LevelBy AUTHOR: 0.0238 / Movie #1: 0.037
Minimum - Maximum-0.08894059 - 0.12200458
Average (Standard dev.)0.00026753 (±0.00716679)
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
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0890.1220.000

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Supplemental data

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Sample components

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Entire : Membrane protein complex ExbB4-ExbD1-TonB1 from Escherichia coli

EntireName: Membrane protein complex ExbB4-ExbD1-TonB1 from Escherichia coliBiological membrane
Components
  • Sample: Membrane protein complex ExbB4-ExbD1-TonB1 from Escherichia coliBiological membrane
  • Protein or peptide: Biopolymer Transport Protein ExbB
  • Protein or peptide: Biopolymer Transport Protein ExbD
  • Protein or peptide: Protein TonB

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Supramolecule #1000: Membrane protein complex ExbB4-ExbD1-TonB1 from Escherichia coli

SupramoleculeName: Membrane protein complex ExbB4-ExbD1-TonB1 from Escherichia coli
type: sample / ID: 1000
Oligomeric state: Four ExbB in complex with one ExbD and one TonB
Number unique components: 3
Molecular weightTheoretical: 150 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 K-12 (bacteria) / Location in cell: Cytoplasmic Membrane
Molecular weightTheoretical: 26 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: pLysS / Recombinant plasmid: pETDuet-1
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: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Location in cell: Cytoplasmic Membrane
Molecular weightTheoretical: 17 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: pLysS / Recombinant plasmid: pETDuet-1
SequenceUniProtKB: Biopolymer transport protein ExbD / GO: membrane => GO:0016020 / InterPro: TonB system transport protein ExbD type-1

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Macromolecule #3: Protein TonB

MacromoleculeName: Protein TonB / type: protein_or_peptide / ID: 3 / Name.synonym: TonB / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Location in cell: Cytoplasmic Membrane
Molecular weightTheoretical: 29 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: pLysS / Recombinant plasmid: pETDuet-1
SequenceUniProtKB: Protein TonB / GO: membrane => GO:0016020 / InterPro: TonB/TolA, C-terminal

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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

BufferpH: 7.5 / Details: 25 mM Tris-HCl, 150 mM NaCl, 0.02% DDM
StainingType: NEGATIVE
Details: Grids with adsorbed protein were stained with 1.5% uranyl formate for 90 seconds.
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: 3.0 µm / Nominal defocus min: 1.0 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
DateApr 22, 2014
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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: Xmipp3.1, RELION1.3 / Number images used: 10900
DetailsManual particle picking from random conical tilt pairs, 3D classification, projection matching angular refinement

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