|Entry||Database: EMDB / ID: EMD-21361|
|Title||TriABC triclosan efflux pump from Pseudomonas aeruginosa- No symmetry|
|Biological species||Pseudomonas aeruginosa (bacteria)|
|Method||single particle reconstruction / cryo EM / Resolution: 6.5 Å|
|Authors||Rouiller I / Fabre L / Bhattacharyya S / Sygusch J|
|Funding support|| Canada, United States, Australia, 6 items |
|Citation||Journal: 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.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_21361.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.5531 Å|
|Symmetry||Space group: 1|
CCP4 map header:
|Entire||Name: TriAxBC / Number of components: 1|
-Component #1: protein, TriAxBC
|Protein||Name: TriAxBC / Recombinant expression: No|
|Mass||Experimental: 560 kDa|
|Source||Species: Pseudomonas aeruginosa (bacteria)|
|Source (engineered)||Expression System: Escherichia coli (E. coli)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Buffer solution: 100 mM Tris HCl (pH 8.0), 150 mM NaCl, 1 mM PMSF, 0.03% (w/v) DDM|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 75000 X (nominal) / Imaging mode: BRIGHT FIELD / Defocus: 1600.0 - 3000.0 nm|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: FEI FALCON II (4k x 4k)|
|Image acquisition||Number of digital images: 3483 |
Details: 1084 images were kept for single particle analysis after exclusion of images that had no graphene oxide and proteins.
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 35520|
|3D reconstruction||Algorithm: BACK PROJECTION / Software: RELION / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF|
-Atomic model buiding
|Modeling #1||Refinement space: REAL|
Details: Subunits 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 ...Details: Subunits 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.
Input PDB model: 3NE5, 3LNN, 2V4D
Chain ID: A, M
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