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- EMDB-48235: Structure of zebrafish OTOP1 in nanodisc in complex with inhibito... -

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Basic information

Entry
Database: EMDB / ID: EMD-48235
TitleStructure of zebrafish OTOP1 in nanodisc in complex with inhibitor C2.36
Map dataSharpened map for zebrafish OTOP1 in nanodisc in complex with inhibitor C2.36
Sample
  • Complex: zebrafish OTOP1 in MSP2N2 nanodisc
    • Protein or peptide: Proton channel OTOP1
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: CHOLESTEROL
  • Ligand: 4-[(4R)-5H,11H-[1,2,4]triazolo[3,4-c][1,4]benzoxazepin-3-yl]phenol
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: 3.42 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_48235.png

Downloads & links

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Map

FileDownload / File: emd_48235.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map for zebrafish OTOP1 in nanodisc in complex with inhibitor C2.36
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 200 pix.
= 208.8 Å		1.04 Å/pix.
x 200 pix.
= 208.8 Å		1.04 Å/pix.
x 200 pix.
= 208.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.044 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.139
Minimum - Maximum-1.7351253 - 2.3682055
Average (Standard dev.)0.0011109055 (±0.040584292)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 208.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_48235_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 zebrafish OTOP1 in nanodisc...

Fileemd_48235_half_map_1.map
AnnotationHalf map B for zebrafish OTOP1 in nanodisc in complex with inhibitor C2.36
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A for zebrafish OTOP1 in nanodisc...

Fileemd_48235_half_map_2.map
AnnotationHalf map A for zebrafish OTOP1 in nanodisc in complex with inhibitor C2.36
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: CHOLESTEROL
  • Ligand: 4-[(4R)-5H,11H-[1,2,4]triazolo[3,4-c][1,4]benzoxazepin-3-yl]phenol

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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: 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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Macromolecule #4: 4-[(4R)-5H,11H-[1,2,4]triazolo[3,4-c][1,4]benzoxazepin-3-yl]phenol

MacromoleculeName: 4-[(4R)-5H,11H-[1,2,4]triazolo[3,4-c][1,4]benzoxazepin-3-yl]phenol
type: ligand / ID: 4 / Number of copies: 6 / Formula: A1BKT
Molecular weightTheoretical: 279.293 Da

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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

Concentration5.3 mg/mL
BufferpH: 8
Component:
ConcentrationName
25.0 mMTris
150.0 mMNaCl
2.0 mMdithiothreitol
5.0 %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 C2.36, reaching a final concentration of 1 mM, and 5% DMSO

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 3 / Number real images: 26378 / Average exposure time: 3.5 sec. / Average electron dose: 45.0 e/Å2
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: 190000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN

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Image processing

DetailsMicrographs with CTF estimates (cryoSPARC Live) worse than 5A were discarded.
Particle selectionNumber selected: 7236014
CTF correctionType: 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: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF
Details: final reported resolution of 3.42A was obtained through the Remote 3DFSC Processing server
Number images used: 290491
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 2 / Avg.num./class: 145000
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-9mfm:
Structure of zebrafish OTOP1 in nanodisc in complex with inhibitor C2.36

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