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- EMDB-9360: Structure of zebrafish Otop1 in nanodiscs -

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Basic information

Entry
Database: EMDB / ID: EMD-9360
TitleStructure of zebrafish Otop1 in nanodiscs
Map dataStructure of zfOtop1 in lipidic nanodiscs
Sample
  • Complex: Zebrafish Otopetrin1 in lipidic nanodiscs
    • Protein or peptide: Otopetrin1
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: CHOLESTEROL
KeywordsProton channel / Otopetrin / MEMBRANE PROTEIN
Biological speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsSaotome K / Lee WH
Funding support United States, 2 items
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, 2018-
Header (metadata) releaseFeb 20, 2019-
Map releaseJun 5, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.41
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 5.41
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6nf4
  • Surface level: 5.41
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9360.png

Downloads & links

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Map

FileDownload / File: emd_9360.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of zfOtop1 in lipidic nanodiscs
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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.41 / Movie #1: 5.41
Minimum - Maximum-22.402294000000001 - 33.984046999999997
Average (Standard dev.)0.000000000012908 (±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-22.40233.9840.000

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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
Components
  • Complex: Zebrafish Otopetrin1 in lipidic nanodiscs
    • Protein or peptide: Otopetrin1
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: CHOLESTEROL

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Supramolecule #1: Zebrafish Otopetrin1 in lipidic nanodiscs

SupramoleculeName: Zebrafish Otopetrin1 in lipidic nanodiscs / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 65853 kDa/nm

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Macromolecule #1: Otopetrin1

MacromoleculeName: Otopetrin1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 65.817 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPVEHGGTDS MWLNKYNPAA ASSASSSSSS DAENKLFSRL KVSLTKKYPQ KNAELLSAQY GTNLLLLGVS VMLALAAQSG PVKEEHLLS FITVLMLVQL VWMLCYMIRR ERERSPVPER DAHAGASWIR GGLTMLALLS LIMDAFRIGY FVGYHSCISA A LGVYPIVH ...String:
GPVEHGGTDS MWLNKYNPAA ASSASSSSSS DAENKLFSRL KVSLTKKYPQ KNAELLSAQY GTNLLLLGVS VMLALAAQSG PVKEEHLLS FITVLMLVQL VWMLCYMIRR ERERSPVPER DAHAGASWIR GGLTMLALLS LIMDAFRIGY FVGYHSCISA A LGVYPIVH ALHTISQVHF LWFHIKDVIK KYETFERFGV IHAVFTNLLL WCNGVMSETE HFMHNHRRRL IEMGYANLST VD VQPHCNC TTSVCSMFST SLYYLYPFNI EYHIFVSAML FVMWKNIGRT LDRHSNRKRR STGSTGLLLG PLGGLVALAS SVS VLVVYL IHLEKTEEMH EAAVSMFYYY GVAMMACMCV GSGTGLLVYR MENRPMDTGS NPARTLDTEL LLASSLGSWL MSWC SVVAS VAEAGQKSPS FSWTSLTYSL LLVLEKCIQN LFIVESLYRR HSEEEEDAAA PQVFSVAVPP YDGILNHGYE AHDKH REAE PAAGSHALSR KQPDAPLPAG QRLDVTPGRK RQILKNICMF LFMCNISLWI LPAFGCRPQY DNPLENETFG TSVWTT VLN VAIPLNLFYR MHSVASLFEV FRKV

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Macromolecule #2: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 2 / Number of copies: 6 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #3: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 3 / Number of copies: 2 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.1 mg/mL
BufferpH: 8
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: ab initio reconstruction in cryosparc
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 67425
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 0.6.5)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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