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- EMDB-5915: The structure of a complete multidrug efflux pump -

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Basic information

Entry
Database: EMDB / ID: EMD-5915
TitleThe structure of a complete multidrug efflux pump
Map dataReconstruction of complete multidrug efflux pump
Sample
  • Sample: AcrB-AcrA-TolC-AcrZ multidrug efflux pump
  • Protein or peptide: AcrABZ-TolC complex
Keywordsmultidrug efflux pump
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsDu D / Wang Z / James NR / Voss JE / Klimont E / Ohene-Agyei T / Venter H / Chiu W / Luisi BF
CitationJournal: Nature / Year: 2014
Title: Structure of the AcrAB-TolC multidrug efflux pump.
Authors: Dijun Du / Zhao Wang / Nathan R James / Jarrod E Voss / Ewa Klimont / Thelma Ohene-Agyei / Henrietta Venter / Wah Chiu / Ben F Luisi /
Abstract: The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of ...The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent 'pumps' that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component. A model system for such a pump is the acridine resistance complex of Escherichia coli. This pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB-TolC efflux pump is able to transport vectorially a diverse array of compounds with little chemical similarity, thus conferring resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including in animal and plant pathogens. Crystal structures are available for the individual components of the pump and have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. However, how the subunits are organized in the pump, their stoichiometry and the details of their interactions are not known. Here we present the pseudo-atomic structure of a complete multidrug efflux pump in complex with a modulatory protein partner from E. coli. The model defines the quaternary organization of the pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species.
History
DepositionMar 1, 2014-
Header (metadata) releaseMar 12, 2014-
Map releaseApr 30, 2014-
UpdateMay 21, 2014-
Current statusMay 21, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5915.gif

Downloads & links

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Map

FileDownload / File: emd_5915.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of complete multidrug efflux pump
Voxel sizeX=Y=Z: 2.47 Å
Density
Contour LevelBy AUTHOR: 7.0 / Movie #1: 7
Minimum - Maximum-10.050803180000001 - 25.346019739999999
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 632.32 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.472.472.47
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z632.320632.320632.320
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-10.05125.346-0.000

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Supplemental data

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Sample components

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Entire : AcrB-AcrA-TolC-AcrZ multidrug efflux pump

EntireName: AcrB-AcrA-TolC-AcrZ multidrug efflux pump
Components
  • Sample: AcrB-AcrA-TolC-AcrZ multidrug efflux pump
  • Protein or peptide: AcrABZ-TolC complex

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Supramolecule #1000: AcrB-AcrA-TolC-AcrZ multidrug efflux pump

SupramoleculeName: AcrB-AcrA-TolC-AcrZ multidrug efflux pump / type: sample / ID: 1000
Oligomeric state: One homotrimer of TolC and one homotrimer of AcrB bound to a homohexamer of AcrA; three AcrZ molecules are bound to the AcrB
Number unique components: 1
Molecular weightTheoretical: 771 KDa

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Macromolecule #1: AcrABZ-TolC complex

MacromoleculeName: AcrABZ-TolC complex / type: protein_or_peptide / ID: 1 / Name.synonym: acridine transport pump / Number of copies: 1 / Oligomeric state: hetero-pentadecamer (15-mer) / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: W3110
Molecular weightTheoretical: 770 KDa
Recombinant expressionOrganism: Escherichia coli. / Recombinant strain: C43 (DE3) / Recombinant plasmid: pET21a and pRSFDuet-1

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.0 mg/mL
BufferpH: 7.5 / Details: 50 mM HEPES pH 7.5, 400 mM NaCl, 0.03% DDM
GridDetails: 200 mesh copper grid with holey carbon film
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: Blot 1 second at force 0

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Electron microscopy

MicroscopeJEOL 3200FSC
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 20000
Specialist opticsEnergy filter - Name: JEOL
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER
TemperatureMin: 78 K / Max: 80 K / Average: 79 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100k magnification
DateMay 20, 2013
Image recordingCategory: CCD / Film or detector model: DIRECT ELECTRON DE-12 (4k x 3k) / Digitization - Sampling interval: 6 µm / Number real images: 1281 / Average electron dose: 25 e/Å2

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Image processing

CTF correctionDetails: per image
Final reconstructionResolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Software - Name: EMAN2, RELION
Details: Resolution was estimated by gold standard eotest from two independent reconstructions.
Number images used: 7000
DetailsParticle were selected by eman2 e2boxer. Initial model was generated by eman2 base on reference free 2d class averages. Further refinements were done by eman2.07, followed by Relion at the end.

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