|Entry||Database: EMDB / ID: EMD-9361|
|Title||Structure of chicken Otop3 in nanodiscs|
|Sample||Zebrafish Otopetrin1 in lipidic nanodiscs:|
Otopetrin3 / ligand
|Biological species||Danio rerio (zebrafish) / Gallus gallus (chicken)|
|Method||single particle reconstruction / cryo EM / Resolution: 3.32 Å|
|Authors||Saotome K / Lee WH / Liman ER / Ward AB|
|Citation||Journal: Nat. Struct. Mol. Biol. / Year: 2019|
Title: Structures of the otopetrin proton channels Otop1 and Otop3.
Authors: Kei Saotome / Bochuan Teng / Che Chun Alex Tsui / Wen-Hsin Lee / Yu-Hsiang Tu / Joshua P Kaplan / Mark S P Sansom / Emily R Liman / Andrew B Ward /
Abstract: Otopetrins (Otop1-Otop3) comprise one of two known eukaryotic proton-selective channel families. Otop1 is required for otoconia formation and a candidate mammalian sour taste receptor. Here we report ...Otopetrins (Otop1-Otop3) comprise one of two known eukaryotic proton-selective channel families. Otop1 is required for otoconia formation and a candidate mammalian sour taste receptor. Here we report cryo-EM structures of zebrafish Otop1 and chicken Otop3 in lipid nanodiscs. The structures reveal a dimeric architecture, with each subunit forming 12 transmembrane helices divided into structurally similar amino (N) and carboxy (C) domains. Cholesterol-like molecules occupy various sites in Otop1 and Otop3 and occlude a central tunnel. In molecular dynamics simulations, hydrophilic vestibules formed by the N and C domains and in the intrasubunit interface between N and C domains form conduits for water entry into the membrane core, suggesting three potential proton conduction pathways. By mutagenesis, we tested the roles of charged residues in each putative permeation pathway. Our results provide a structural basis for understanding selective proton permeation and gating of this conserved family of proton channels.
|Validation Report||PDB-ID: 6nf6|
SummaryFull reportAbout validation report
|Date||Deposition: Dec 18, 2018 / Header (metadata) release: Feb 20, 2019 / Map release: Jun 5, 2019 / Update: Jun 19, 2019|
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_9361.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.03 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Zebrafish Otopetrin1 in lipidic nanodiscs
|Entire||Name: Zebrafish Otopetrin1 in lipidic nanodiscs / Number of components: 3|
-Component #1: protein, Zebrafish Otopetrin1 in lipidic nanodiscs
|Protein||Name: Zebrafish Otopetrin1 in lipidic nanodiscs / Recombinant expression: No|
|Source||Species: Danio rerio (zebrafish)|
|Source (engineered)||Expression System: Homo sapiens (human)|
-Component #2: protein, Otopetrin3
|Protein||Name: Otopetrin3 / Number of Copies: 2 / Recombinant expression: No|
|Mass||Theoretical: 63.744496 kDa|
|Source||Species: Gallus gallus (chicken)|
|Source (engineered)||Expression System: Homo sapiens (human)|
-Component #3: ligand, CHOLESTEROL HEMISUCCINATE
|Ligand||Name: CHOLESTEROL HEMISUCCINATE / Number of Copies: 2 / Recombinant expression: No|
|Mass||Theoretical: 0.486726 kDa|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 2.1 mg/mL / pH: 8|
|Vitrification||Cryogen name: ETHANE / Temperature: 283 K / Humidity: 100 %|
-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||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Processing||Method: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 43667|
|3D reconstruction||Software: RELION / Resolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF|
|FSC plot (resolution estimation)|
-Atomic model buiding
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