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- PDB-6nf6: Structure of chicken Otop3 in nanodiscs -

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Basic information

Entry
Database: PDB / ID: 6nf6
TitleStructure of chicken Otop3 in nanodiscs
ComponentsOtopetrin3
KeywordsMEMBRANE PROTEIN / Proton channel / Otopetrin
Function / homologyCHOLESTEROL HEMISUCCINATE
Function and homology information
Biological speciesGallus gallus (chicken)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsSaotome, K. / Lee, W.H. / Liman, E.R. / Ward, A.B.
Funding support United States, 2items
OrganizationGrant numberCountry
Other private United States
National Institutes of Health/National Institute on Deafness and Other Communication Disorders (NIH/NIDCD)NIDCD013741 United States
CitationJournal: 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.
History
DepositionDec 18, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 5, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.funding_organization
Revision 1.3Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Otopetrin3
B: Otopetrin3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)128,4624
Polymers127,4892
Non-polymers9732
Water0
1


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

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Components

#1: Protein Otopetrin3


Mass: 63744.496 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#2: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C31H50O4

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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: Zebrafish Otopetrin1 in lipidic nanodiscs / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 65853 kDa/nm / Experimental value: NO
Source (natural)Organism: Danio rerio (zebrafish)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenConc.: 2.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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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
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION2.1particle selection
4GctfCTF correction
7Coot0.8.8model fitting
9cryoSPARC0.6.5initial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
13PHENIX1.14model refinement
14Rosettamodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43667 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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