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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 data
SampleE.coli cell overexpressing AcrAB-TolC treated with AcrB inhibitor
Biological speciesEscherichia coli BL21(DE3) (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 20 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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Structure viewerEM map:
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Supplemental images

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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.78 Å/pix.
x 160 pix.
= 444.8 Å
2.78 Å/pix.
x 160 pix.
= 444.8 Å
2.78 Å/pix.
x 160 pix.
= 444.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

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
Number of components: 1

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Component #1: cellular-component, E.coli cell overexpressing AcrAB-TolC treated...

Cellular-componentName: E.coli cell overexpressing AcrAB-TolC treated with AcrB inhibitor
Recombinant expression: No
SourceSpecies: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Cell / Method: cryo EM
Sample solutionpH: 7
Support filmunspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

ImagingMicroscope: JEOL 3200FSC
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 3 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: subtomogram averaging / Applied symmetry: C3 (3 fold cyclic) / Number of subtomograms: 678
3D reconstructionAlgorithm: FOURIER SPACE / Software: EMAN2 / Resolution: 20 Å / Resolution method: FSC 0.143 CUT-OFF / Euler angles: Subtomogram alignment
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body
Input PDB model: 5V5S

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