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- EMDB-21363: Cryo-EM map of TriABC triclosan efflux pump from Pseudomonas aeru... -

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Basic information

Entry
Database: EMDB / ID: EMD-21363
TitleCryo-EM map of TriABC triclosan efflux pump from Pseudomonas aeruginosa
Map dataCryoEM map of the TriABC triclosan efflux pump from Pseudomonas aeruginosa, C3 imposed
Sample
  • Complex: TriAxBC
Function / homology
Function and homology information


efflux pump complex / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / transmembrane transporter activity / membrane => GO:0016020 / cell division / plasma membrane
Similarity search - Function
RND efflux pump, membrane fusion protein / RND efflux pump, membrane fusion protein, barrel-sandwich domain / Barrel-sandwich domain of CusB or HlyD membrane-fusion / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family
Similarity search - Domain/homology
Probable Resistance-Nodulation-Cell Division (RND) efflux transporter / Probable Resistance-Nodulation-Cell Division (RND) efflux membrane fusion protein / Probable Resistance-Nodulation-Cell Division (RND) efflux membrane fusion protein
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsFabre L / Bhattacharyya S / Ruickoldt J / Rouiller I / Zgurskaya HI / 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: 2021
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 releaseSep 30, 2020-
UpdateDec 23, 2020-
Current statusDec 23, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.158
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.158
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vej
  • Surface level: 0.15
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21363.png

Downloads & links

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Map

FileDownload / File: emd_21363.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM map of the TriABC triclosan efflux pump from Pseudomonas aeruginosa, C3 imposed
Voxel sizeX=Y=Z: 1.5531 Å
Density
Contour LevelBy AUTHOR: 0.158 / Movie #1: 0.158
Minimum - Maximum-0.2711445 - 0.598383
Average (Standard dev.)0.0067000864 (±0.029969608)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 217.434 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.55311.55311.5531
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z217.434217.434217.434
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS140140140
D min/max/mean-0.2710.5980.007

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Supplemental data

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Sample components

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Entire : TriAxBC

EntireName: TriAxBC
Components
  • Complex: TriAxBC

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Supramolecule #1: TriAxBC

SupramoleculeName: TriAxBC / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 560 KDa

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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

BufferpH: 8
Details: 100 mM Tris HCl (pH 8.0), 150 mM NaCl, 1 mM PMSF, 0.03% (w/v) DDM
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Number grids imaged: 1 / Number real images: 3483 / Average electron dose: 50.0 e/Å2
Details: 1084 images were kept for single particle analysis after exclusion of images that had no graphene oxide and proteins.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 35520
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: EMDB MAP / Details: EMD-21362
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 35520

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Atomic model buiding 1

Initial model
PDB IDChain

3ne5

residue_range: 1-1016

3lnn

chain_id: A

2v4d

chain_id: M
DetailsSubunits A-C were modeled with I-TASSER using CusA (3NE5) and then fitted into the EM density and refined with Phenix. Model was rebuilt using Coot. The N-terminal regions of subunits P-R were modeled with I-TASSER based on MP domain of 3LNN while C-terminal regions of subunits P-R were modelled based on MP domain of 2V4D and similarly refined with model rebuilding.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6vej:
TriABC transporter from Pseudomonas aeruginosa

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