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- PDB-6o84: Cryo-EM structure of OTOP3 from xenopus tropicalis -

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Entry
Database: PDB / ID: 6o84
TitleCryo-EM structure of OTOP3 from xenopus tropicalis
ComponentsLOC100127796 protein,LOC100127796 protein,OTOP3,LOC100127796 protein,LOC100127796 protein
KeywordsMEMBRANE PROTEIN / Otopetrin / proton / channel
Function / homologyOtopetrin / Otopetrin / monoatomic ion transport / membrane => GO:0016020 / plasma membrane / LOC100127796 protein
Function and homology information
Biological speciesXenopus tropicalis (tropical clawed frog)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.92 Å
AuthorsChen, Q.F. / Bai, X.C. / Jiang, Y.X.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR)GM079179 United States
Howard Hughes Medical Institute (HHMI) United States
Other privatethe Cancer Prevention and Research Initiative of Texas United States
Other privateMurchison Linthicum Scholar in Medical Research fund United States
Welch FoundationI-1578 United States
CitationJournal: Elife / Year: 2019
Title: Structural and functional characterization of an otopetrin family proton channel.
Authors: Qingfeng Chen / Weizhong Zeng / Ji She / Xiao-Chen Bai / Youxing Jiang /
Abstract: The otopetrin (OTOP) proteins were recently characterized as proton channels. Here we present the cryo-EM structure of OTOP3 from (XtOTOP3) along with functional characterization of the channel. ...The otopetrin (OTOP) proteins were recently characterized as proton channels. Here we present the cryo-EM structure of OTOP3 from (XtOTOP3) along with functional characterization of the channel. XtOTOP3 forms a homodimer with each subunit containing 12 transmembrane helices that can be divided into two structurally homologous halves; each half assembles as an α-helical barrel that could potentially serve as a proton conduction pore. Both pores open from the extracellular half before becoming occluded at a central constriction point consisting of three highly conserved residues - Gln-Asp/Asn-Tyr (the constriction triads). Mutagenesis shows that the constriction triad from the second pore is less amenable to perturbation than that of the first pore, suggesting an unequal contribution between the two pores to proton transport. We also identified several key residues at the interface between the two pores that are functionally important, particularly Asp509, which confers intracellular pH-dependent desensitization to OTOP channels.
History
DepositionMar 8, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
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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Structure visualization

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Assembly

Deposited unit
A: LOC100127796 protein,LOC100127796 protein,OTOP3,LOC100127796 protein,LOC100127796 protein
B: LOC100127796 protein,LOC100127796 protein,OTOP3,LOC100127796 protein,LOC100127796 protein


Theoretical massNumber of molelcules
Total (without water)146,7052
Polymers146,7052
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3370 Å2
ΔGint-45 kcal/mol
Surface area42260 Å2
MethodPISA

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Components

#1: Protein LOC100127796 protein,LOC100127796 protein,OTOP3,LOC100127796 protein,LOC100127796 protein


Mass: 73352.297 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus tropicalis (tropical clawed frog)
Gene: otop3, LOC100127796 / Production host: Homo sapiens (human) / References: UniProt: A9JTM7

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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: Xenopus tropicalis OTOP3 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.077 MDa / Experimental value: NO
Source (natural)Organism: Xenopus tropicalis (tropical clawed frog)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F / Plasmid: pEZT
Buffer solutionpH: 8
Details: Solutions were made fresh from stock solutions and filtered.
Buffer componentConc.: 150 mM / Name: sodium chloride / Formula: NaClSodium chloride
SpecimenConc.: 1.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 / Nominal magnification: 46730 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3000
EM imaging opticsEnergyfilter name: GIF Quantum LS
Image scansMovie frames/image: 30

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
10RELION2initial Euler assignment
11RELION2final Euler assignment
12RELION2classification
13RELION23D reconstruction
Image processingDetails: 30 frames per movie stack were saved for motion correction.
CTF correctionDetails: The CTF correction was performed during the map refinement in RELION.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1433949 / Details: The particles were auto-picked in RELION.
3D reconstructionResolution: 3.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 212551 / Symmetry type: POINT

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