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- EMDB-0533: AcrAB-TolC open state - In situ structure and assembly of multidr... -

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Basic information

Entry
Database: EMDB / ID: EMD-0533
TitleAcrAB-TolC open state - In situ structure and assembly of multidrug efflux pump AcrAB-TolC
Map dataAveraged structure of TolC-AcrAB in e.coli cellular tomogram with AcrB inhibitor.
Sample
  • Cell: E.coli cell overexpressing AcrAB-TolC treated with AcrB inhibitor
Biological speciesEscherichia coli BL21(DE3) (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 20.0 Å
AuthorsChen M / Shi X / Ludtke SJ / Wang Z
Funding support United States, 2 items
OrganizationGrant numberCountry
Welch FoundationQ-1967-20180324 United States
National Institutes of Health/National Institute of General Medical SciencesR01GM080139 United States
CitationJournal: Nat Commun / Year: 2019
Title: In situ structure and assembly of the multidrug efflux pump AcrAB-TolC.
Authors: Xiaodong Shi / Muyuan Chen / Zhili Yu / James M Bell / Hans Wang / Isaac Forrester / Heather Villarreal / Joanita Jakana / Dijun Du / Ben F Luisi / Steven J Ludtke / Zhao Wang /
Abstract: Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form ...Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.
History
DepositionFeb 7, 2019-
Header (metadata) releaseMar 27, 2019-
Map releaseJun 26, 2019-
UpdateJun 26, 2019-
Current statusJun 26, 2019Processing site: RCSB / 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

emd_0533.png

Downloads & links

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Map

FileDownload / File: emd_0533.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAveraged structure of TolC-AcrAB in e.coli cellular tomogram with AcrB inhibitor.
Voxel sizeX=Y=Z: 2.78 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-2.083301 - 2.7269228
Average (Standard dev.)0.02340048 (±0.15093714)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-80-80
Dimensions160160160
Spacing160160160
CellA=B=C: 444.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.782.782.78
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z444.800444.800444.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ304304304
MAP C/R/S123
start NC/NR/NS-80-80-80
NC/NR/NS160160160
D min/max/mean-2.0832.7270.023

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Supplemental data

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Sample components

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Entire : E.coli cell overexpressing AcrAB-TolC treated with AcrB inhibitor

EntireName: E.coli cell overexpressing AcrAB-TolC treated with AcrB inhibitor
Components
  • Cell: E.coli cell overexpressing AcrAB-TolC treated with AcrB inhibitor

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Supramolecule #1: E.coli cell overexpressing AcrAB-TolC treated with AcrB inhibitor

SupramoleculeName: E.coli cell overexpressing AcrAB-TolC treated with AcrB inhibitor
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7
GridDetails: unspecified
VitrificationCryogen name: ETHANE
DetailsTreated with MBX3132

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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: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 3.0 e/Å2

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Image processing

ExtractionNumber tomograms: 12 / Number images used: 678 / Software - Name: EMAN2 (ver. 2.3)
CTF correctionSoftware - Name: EMAN2 (ver. 2.3)
Final angle assignmentType: OTHER / Software - Name: EMAN2 (ver. 2.3) / Details: Subtomogram alignment
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 (ver. 2.3) / Number subtomograms used: 678
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5v5s

RefinementProtocol: RIGID BODY FIT

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