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- EMDB-48234: Structure of zebrafish OTOP1 in nanodisc in the presence of inhib... -

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Basic information

Entry
Database: EMDB / ID: EMD-48234
TitleStructure of zebrafish OTOP1 in nanodisc in the presence of inhibitor C11
Map dataRefinement map for structure of zebrafish OTOP1 in nanodisc in the presence of inhibitor C11
Sample
  • Complex: zebrafish OTOP1 in MSP2N2 nanodisc
    • Protein or peptide: Proton channel OTOP1
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: (3beta,5beta,14beta,17alpha)-cholestan-3-ol
Keywordsproton channel / ion channel / OTOP / Otopetrin / membrane protein
Function / homology
Function and homology information


otolith formation / otolith mineralization / proton channel activity / inner ear morphogenesis / cholesterol binding / negative regulation of type II interferon-mediated signaling pathway / microvillus / proton transmembrane transport / cellular response to insulin stimulus / identical protein binding ...otolith formation / otolith mineralization / proton channel activity / inner ear morphogenesis / cholesterol binding / negative regulation of type II interferon-mediated signaling pathway / microvillus / proton transmembrane transport / cellular response to insulin stimulus / identical protein binding / membrane / plasma membrane
Similarity search - Function
Proton channel OTOP1
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.73 Å
AuthorsBurendei B / Ward AB
Funding support United States, 1 items
OrganizationGrant numberCountry
Not funded United States
CitationJournal: Nat Commun / Year: 2025
Title: Structure-guided discovery of Otopetrin 1 inhibitors reveals druggable binding sites at the intrasubunit interface.
Authors: Batuujin Burendei / Joshua P Kaplan / Gerardo M Orellana / Emily R Liman / Stefano Forli / Andrew B Ward /
Abstract: Proton conductance across cell membranes serves many biological functions, ranging from the regulation of intracellular and extracellular pH to the generation of electrical signals that lead to ...Proton conductance across cell membranes serves many biological functions, ranging from the regulation of intracellular and extracellular pH to the generation of electrical signals that lead to sour taste perception. Otopetrins (OTOPs) are a conserved, eukaryotic family of proton-selective ion channels, one of which (OTOP1) serves as a gustatory sensor for sour tastes and ammonium chloride. As the functional properties and structures of OTOP channels were only recently described, there are presently few tools available to modulate their activity. Here, we perform subsequent rounds of molecular docking-based virtual screening against the structure of zebrafish OTOP1, followed by functional testing using whole-cell patch-clamp electrophysiology, and identify several small molecule inhibitors that are effective in the low-to-mid µM range. Cryo-electron microscopy structures reveal inhibitor binding sites in the intrasubunit interface that are validated by functional testing of mutant channels. Our findings reveal pockets that can be targeted for small molecule discovery to develop modulators for Otopetrins. Such modulators can serve as useful toolkit molecules for future investigations of structure-function relationships or physiological roles of Otopetrins.
History
DepositionDec 10, 2024-
Header (metadata) releaseNov 5, 2025-
Map releaseNov 5, 2025-
UpdateNov 5, 2025-
Current statusNov 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48234.png

Downloads & links

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Map

FileDownload / File: emd_48234.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefinement map for structure of zebrafish OTOP1 in nanodisc in the presence of inhibitor C11
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 256 pix.
= 294.4 Å		1.15 Å/pix.
x 256 pix.
= 294.4 Å		1.15 Å/pix.
x 256 pix.
= 294.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.24
Minimum - Maximum-1.4509318 - 2.2173505
Average (Standard dev.)-0.00021568108 (±0.034182683)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 294.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_48234_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B for structure of zebrafish OTOP1...

Fileemd_48234_half_map_1.map
AnnotationHalf map B for structure of zebrafish OTOP1 in nanodisc in the presence of inhibitor C11
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A for structure of zebrafish OTOP1...

Fileemd_48234_half_map_2.map
AnnotationHalf map A for structure of zebrafish OTOP1 in nanodisc in the presence of inhibitor C11
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : zebrafish OTOP1 in MSP2N2 nanodisc

EntireName: zebrafish OTOP1 in MSP2N2 nanodisc
Components
  • Complex: zebrafish OTOP1 in MSP2N2 nanodisc
    • Protein or peptide: Proton channel OTOP1
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: (3beta,5beta,14beta,17alpha)-cholestan-3-ol

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Supramolecule #1: zebrafish OTOP1 in MSP2N2 nanodisc

SupramoleculeName: zebrafish OTOP1 in MSP2N2 nanodisc / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 131 KDa

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Macromolecule #1: Proton channel OTOP1

MacromoleculeName: Proton channel OTOP1 / type: protein_or_peptide / ID: 1
Details: first two residues (GP) are leftover residues from a 3C protease cleavage site. 3rd residue (V) corresponds to the 2nd residue of the zebrafish OTOP1 sequence (Uniprot:Q7ZWK8)
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

UniProtKB: Proton channel OTOP1

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Macromolecule #2: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 2 / Number of copies: 4 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #3: (3beta,5beta,14beta,17alpha)-cholestan-3-ol

MacromoleculeName: (3beta,5beta,14beta,17alpha)-cholestan-3-ol / type: ligand / ID: 3 / Number of copies: 4 / Formula: QNJ
Molecular weightTheoretical: 388.669 Da
Chemical component information

ChemComp-QNJ:
(3beta,5beta,14beta,17alpha)-cholestan-3-ol

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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 mg/mL
BufferpH: 8
Component:
ConcentrationName
25.0 mMTris
150.0 mMNaCl
2.0 mMdithiothreitol
1.0 %dimethylsulfoxide
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV
Detailssample was complexed with small molecule C11, reaching a final concentration of 1.11mM, and 1% DMSO

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Number grids imaged: 1 / Number real images: 3552 / Average exposure time: 9.8 sec. / Average electron dose: 50.37 e/Å2
Details: Forty-nine frames of 200 ms exposure time were collected per movie
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

DetailsMicrographs with CTF estimates (Gctf) worse than 5A were discarded.
Particle selectionNumber selected: 3410686
CTF correctionSoftware - Name: Gctf (ver. 1.06) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: EMDB MAP
EMDB ID:

9360

Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.0)
Details: final reported resolution of 3.73A was obtained through the Remote 3DFSC Processing server
Number images used: 164766
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.0)
Final 3D classificationNumber classes: 3 / Avg.num./class: 70000 / Software - Name: cryoSPARC (ver. 3.3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6nf4


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-9mfl:
Structure of zebrafish OTOP1 in nanodisc in the presence of inhibitor C11

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