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- EMDB-6318: Three-dimensional structure of AcrAB and MacA-TolC alpha-hybrid d... -

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Basic information

Entry
Database: EMDB / ID: EMD-6318
TitleThree-dimensional structure of AcrAB and MacA-TolC alpha-hybrid dimer complex
Map dataReconstruction of negatively stained AcrB-transmembrane linker-AcrA-AcrA and MacA-TolC alpha hybrid dimer complex, which supports direct interaction between AcrA and TolC
Sample
  • Sample: AcrB-TM#5-AcrA-AcrA and MacA-TolC alpha hybrid dimer complex
  • Protein or peptide: AcrAB and MacA-TolC alpha hybrid dimer complex
Keywordsmultidrug efflux pump / multidrug resistance / resistance-nodulation-division family
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 24.0 Å
AuthorsKim J-S / Jeong H / Song S / Kim H-Y / Lee K / Hyun J / Ha N-C
CitationJournal: Mol Cells / Year: 2015
Title: Structure of the tripartite multidrug efflux pump AcrAB-TolC suggests an alternative assembly mode.
Authors: Jin-Sik Kim / Hyeongseop Jeong / Saemee Song / Hye-Yeon Kim / Kangseok Lee / Jaekyung Hyun / Nam-Chul Ha /
Abstract: Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation ...Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the α-barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria.
History
DepositionApr 15, 2015-
Header (metadata) releaseAug 26, 2015-
Map releaseAug 26, 2015-
UpdateMay 25, 2016-
Current statusMay 25, 2016Processing site: PDBj / 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 cylindrical radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6318.gif

Downloads & links

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Map

FileDownload / File: emd_6318.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of negatively stained AcrB-transmembrane linker-AcrA-AcrA and MacA-TolC alpha hybrid dimer complex, which supports direct interaction between AcrA and TolC
Voxel sizeX=Y=Z: 2.1 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-3.90563107 - 3.46254587
Average (Standard dev.)-0.00001689 (±0.17771661)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 537.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.12.12.1
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z537.600537.600537.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-3.9063.463-0.000

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Supplemental data

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Sample components

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Entire : AcrB-TM#5-AcrA-AcrA and MacA-TolC alpha hybrid dimer complex

EntireName: AcrB-TM#5-AcrA-AcrA and MacA-TolC alpha hybrid dimer complex
Components
  • Sample: AcrB-TM#5-AcrA-AcrA and MacA-TolC alpha hybrid dimer complex
  • Protein or peptide: AcrAB and MacA-TolC alpha hybrid dimer complex

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Supramolecule #1000: AcrB-TM#5-AcrA-AcrA and MacA-TolC alpha hybrid dimer complex

SupramoleculeName: AcrB-TM#5-AcrA-AcrA and MacA-TolC alpha hybrid dimer complex
type: sample / ID: 1000
Oligomeric state: AcrB trimer, AcrA hexamer and MacA-TolC trimer
Number unique components: 1
Molecular weightTheoretical: 700 KDa

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Macromolecule #1: AcrAB and MacA-TolC alpha hybrid dimer complex

MacromoleculeName: AcrAB and MacA-TolC alpha hybrid dimer complex / type: protein_or_peptide / ID: 1
Details: AcrAB fusion protein was generated by fusing functional AcrA dimer to the C-terminus of AcrB with a short transmembrane linker, and MacA-TolC alpha hybrid dimer was bound to the fusion ...Details: AcrAB fusion protein was generated by fusing functional AcrA dimer to the C-terminus of AcrB with a short transmembrane linker, and MacA-TolC alpha hybrid dimer was bound to the fusion protein in order to generate the complete tripartite complex
Number of copies: 12
Oligomeric state: Dodecamer (3 x AcrB, 6 x AcrA, 3 x MacA-TolC)
Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET22b

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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: 7.5 / Details: 20mM HEPES, 150mM NaCl, 0.02% DDM, 10% glycerol
StainingType: NEGATIVE
Details: Grid was stained with 0.75% uranyl formate for 60 seconds, followed by blotting of excess solution using a piece of filter paper
GridDetails: 300-mesh copper EM-grid with covered with thin carbon film, glow discharged in air
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 66667 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 6.3 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateMar 10, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 100
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTF correction to each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 1563
DetailsParticles were manually selected and boxed in 256 x 256 pixel boxes using e2boxer.py. Boxed particles were used to estimate and correct for CTF using e2ctf.py. Initial model from selected class averages was generated using e2initialmodel.py, and the 3D refinement was performed using e2refine_easy.py.

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