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- EMDB-21362: Negative stain map of TriABC triclosan efflux pump from Pseudomon... -

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Basic information

Entry
Database: EMDB / ID: EMD-21362
TitleNegative stain map of TriABC triclosan efflux pump from Pseudomonas aeruginosa
Map data
SampleTriAxBC
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 20 Å
AuthorsRouiller I / Fabre L / Sygusch J
Funding support Canada, United States, Australia, 6 items
OrganizationGrant numberCountry
Other governmentFRSQ- GEPROM- Seed Grant Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2016-04898 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2014-04798 Canada
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI052293 United States
Canadian Institutes of Health Research (CIHR)MOP 86693 Canada
Other privateUniversity of Melbourne- Start-up Funds Australia
CitationJournal: Structure / Year: 2020
Title: A "Drug Sweeping" State of the TriABC Triclosan Efflux Pump from Pseudomonas aeruginosa.
Authors: Lucien Fabre / Abigail T Ntreh / Amira Yazidi / Inga V Leus / Jon W Weeks / Sudipta Bhattacharyya / Jakob Ruickoldt / Isabelle Rouiller / Helen I Zgurskaya / Jurgen Sygusch /
Abstract: The structure of the TriABC inner membrane component of the triclosan/SDS-specific efflux pump from Pseudomonas aeruginosa was determined by cryoelectron microscopy to 4.5 Å resolution. The ...The structure of the TriABC inner membrane component of the triclosan/SDS-specific efflux pump from Pseudomonas aeruginosa was determined by cryoelectron microscopy to 4.5 Å resolution. The complete structure of the inner membrane transporter TriC of the resistance-nodulation-division (RND) superfamily was solved, including a partial structure of the fused periplasmic membrane fusion subunits, TriA and TriB. The substrate-free conformation of TriABC represents an intermediate step in efflux complex assembly before the engagement of the outer membrane channel. Structural analysis identified a tunnel network whose constriction impedes substrate efflux, indicating inhibition of TriABC in the unengaged state. Blind docking studies revealed binding to TriC at the same loci by substrates and bulkier non-substrates. Together with functional analyses, we propose that selective substrate translocation involves conformational gating at the tunnel narrowing that, together with conformational ordering of TriA and TriB, creates an engaged state capable of mediating substrate efflux.
History
DepositionFeb 6, 2020-
Header (metadata) releaseMar 4, 2020-
Map releaseOct 28, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0539
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0539
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21362.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.59 Å/pix.
x 140 pix.
= 223.006 Å
1.59 Å/pix.
x 140 pix.
= 223.006 Å
1.59 Å/pix.
x 140 pix.
= 223.006 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5929 Å
Density
Contour LevelBy AUTHOR: 0.0539 / Movie #1: 0.0539
Minimum - Maximum-0.078162216 - 0.209907
Average (Standard dev.)-0.0012258636 (±0.0487202)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 223.00601 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.59291.59291.5929
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z223.006223.006223.006
α/β/γ90.00090.00090.000
start NX/NY/NZ79740
NX/NY/NZ93103213
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS140140140
D min/max/mean-0.0780.210-0.001

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Supplemental data

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Sample components

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Entire TriAxBC

EntireName: TriAxBC / Number of components: 1

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Component #1: protein, TriAxBC

ProteinName: TriAxBC / Recombinant expression: No
MassExperimental: 560 kDa
SourceSpecies: Pseudomonas aeruginosa (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: negative staining
Sample solutionBuffer solution: 100 mM Tris HCl (pH 8.0), 150 mM NaCl, 1 mM PMSF, 0.03% (w/v) DDM
pH: 8
Support filmunspecified
VitrificationCryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 75000 X (nominal) / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 2000.0 nm
Specimen HolderModel: SIDE ENTRY, EUCENTRIC
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C3 (3 fold cyclic) / Number of projections: 11393
3D reconstructionSoftware: RELION / Resolution: 20 Å / Resolution method: FSC 0.143 CUT-OFF

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