|Entry||Database: PDB / ID: 6nf4|
|Title||Structure of zebrafish Otop1 in nanodiscs|
|Keywords||MEMBRANE PROTEIN / Proton channel / Otopetrin|
|Biological species||Danio rerio (zebrafish)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 Å|
|Authors||Saotome, K. / Lee, W.H. / Liman, E.R. / Ward, A.B.|
|Funding support||United States , 2件 |
|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.
SummaryFull reportAbout validation report
|Date||Deposition: Dec 18, 2018 / Release: Jun 5, 2019|
|Structure viewer||Molecule: |
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Mass: 65817.000 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: Zebrafish Otopetrin1 in lipidic nanodiscs / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT|
|Molecular weight||Value: 65853 kDa/nm / Experimental value: NO|
|Source (natural)||Organism: Danio rerio (zebrafish)|
|Source (recombinant)||Organism: Homo sapiens (human)|
|Buffer solution||pH: 8|
|Specimen||Conc.: 2.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid material: GOLD / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3|
|Vitrification||Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Image recording||Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Symmetry||Point symmetry: C2 (2 fold cyclic)|
|3D reconstruction||Resolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67425 / Symmetry type: POINT|
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