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- PDB-6vej: TriABC transporter from Pseudomonas aeruginosa -

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Basic information

Entry
Database: PDB / ID: 6vej
TitleTriABC transporter from Pseudomonas aeruginosa
Components
  • Probable Resistance-Nodulation-Cell Division (RND) efflux membrane fusion protein,Probable Resistance-Nodulation-Cell Division (RND) efflux membrane fusion protein
  • Probable Resistance-Nodulation-Cell Division (RND) efflux transporter
KeywordsTRANSPORT PROTEIN / RND transporter / Pseudomonas aeruginosa / transmembrane / TriABC / TriC / PMF / cryoEM
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
(2R)-2-(hexadecanoyloxy)propyl nonadecanoate / PALMITIC ACID / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsSygusch, J. / Fabre, L. / Rouiller, I. / Bhattacharyya, S.
Funding support Canada, United States, Australia, 6items
OrganizationGrant numberCountry
Other governmentFRSQ- GEPROM- Seed Grant Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2014-04798 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2016-04898 Canada
Canadian Institutes of Health Research (CIHR)MOP 86693 Canada
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI052293 United States
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
DepositionJan 2, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 30, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 28, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Mar 17, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Probable Resistance-Nodulation-Cell Division (RND) efflux transporter
B: Probable Resistance-Nodulation-Cell Division (RND) efflux transporter
C: Probable Resistance-Nodulation-Cell Division (RND) efflux transporter
P: Probable Resistance-Nodulation-Cell Division (RND) efflux membrane fusion protein,Probable Resistance-Nodulation-Cell Division (RND) efflux membrane fusion protein
Q: Probable Resistance-Nodulation-Cell Division (RND) efflux membrane fusion protein,Probable Resistance-Nodulation-Cell Division (RND) efflux membrane fusion protein
R: Probable Resistance-Nodulation-Cell Division (RND) efflux membrane fusion protein,Probable Resistance-Nodulation-Cell Division (RND) efflux membrane fusion protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)565,18615
Polymers561,1006
Non-polymers4,0869
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Probable Resistance-Nodulation-Cell Division (RND) efflux transporter


Mass: 112962.609 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: PA0158 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9I6X4*PLUS
#2: Protein Probable Resistance-Nodulation-Cell Division (RND) efflux membrane fusion protein,Probable Resistance-Nodulation-Cell Division (RND) efflux membrane fusion protein


Mass: 74070.781 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: PA0156, PA0157 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9I6X6, UniProt: Q9I6X5
#3: Sugar ChemComp-LMU / DODECYL-ALPHA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM
#4: Chemical ChemComp-E2V / (2R)-2-(hexadecanoyloxy)propyl nonadecanoate


Mass: 594.992 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C38H74O4
#5: Chemical ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C16H32O2
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: TriABC / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.560 MDa / Experimental value: YES
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Details: 100 mM Tris HCl (pH 8.0), 150 mM NaCl, 1 mM PMSF, 0.03% (w/v) DDM.
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GRAPHENE OXIDE / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1600 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3483
Details: 1084 images kept for single particle analysis after exclusion of images that had no graphene oxide and proteins.

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Processing

EM software
IDNameVersionCategoryDetails
1EMAN2particle selectione2boxer.py
2EPUimage acquisition
4CTFFIND4CTF correction
10RELION1.3initial Euler assignment
11RELION1.3final Euler assignment
13RELION1.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 35530
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35220 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: Subunits A-C were modeled by I-TASSER using CusA (3NE5) and then fitted into the EM density and refined with Phenix and model rebuilt using Coot. The N-terminal regions of subunits P-R were ...Details: Subunits A-C were modeled by I-TASSER using CusA (3NE5) and then fitted into the EM density and refined with Phenix and model rebuilt using Coot. The N-terminal regions of subunits P-R were modeled by 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 and model rebuilt.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
13NE5

3ne5

11-1016
23LNN

3lnn

A1
32V4D

2v4d

M1

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