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- EMDB-9361: Structure of chicken Otop3 in nanodiscs -

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Entry
Database: EMDB / ID: EMD-9361
TitleStructure of chicken Otop3 in nanodiscs
Map data
SampleZebrafish Otopetrin1 in lipidic nanodiscs:
Otopetrin3 / ligand
Biological speciesDanio rerio (zebrafish) / Gallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsSaotome K / Lee WH / Liman ER / Ward AB
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.
Validation ReportPDB-ID: 6nf6

SummaryFull reportAbout validation report
DateDeposition: Dec 18, 2018 / Header (metadata) release: Feb 20, 2019 / Map release: Jun 5, 2019 / Update: Jun 19, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 5.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6nf6
  • Surface level: 5.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_9361.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.03 Å/pix.
x 200 pix.
= 206. Å
1.03 Å/pix.
x 200 pix.
= 206. Å
1.03 Å/pix.
x 200 pix.
= 206. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 5.5 / Movie #1: 5.5
Minimum - Maximum-19.310072000000002 - 26.482925000000002
Average (Standard dev.)0.000000000010935 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 206.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z206.000206.000206.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ200200200
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-19.31026.4830.000

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Supplemental data

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Sample components

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Entire Zebrafish Otopetrin1 in lipidic nanodiscs

EntireName: Zebrafish Otopetrin1 in lipidic nanodiscs / Number of components: 3

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Component #1: protein, Zebrafish Otopetrin1 in lipidic nanodiscs

ProteinName: Zebrafish Otopetrin1 in lipidic nanodiscs / Recombinant expression: No
MassTheoretical: 65.853GDa
SourceSpecies: Danio rerio (zebrafish)
Source (engineered)Expression System: Homo sapiens (human)

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Component #2: protein, Otopetrin3

ProteinName: Otopetrin3 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 63.744496 kDa
SourceSpecies: Gallus gallus (chicken)
Source (engineered)Expression System: Homo sapiens (human)

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Component #3: ligand, CHOLESTEROL HEMISUCCINATE

LigandName: CHOLESTEROL HEMISUCCINATE / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.486726 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2.1 mg/mL / pH: 8
VitrificationCryogen name: ETHANE / Temperature: 283 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 43667
3D reconstructionSoftware: RELION / Resolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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