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- EMDB-0650: Cryo-EM structure of OTOP3 from xenopus tropicalis -

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Basic information

Entry
Database: EMDB / ID: EMD-0650
TitleCryo-EM structure of OTOP3 from xenopus tropicalis
Map dataCryo-EM structure of OTOP3 from xenopus tropicalis
Sample
  • Organelle or cellular component: Xenopus tropicalis OTOP3
    • Protein or peptide: LOC100127796 protein,LOC100127796 protein,OTOP3,LOC100127796 protein,LOC100127796 protein
KeywordsOtopetrin / proton / channel / MEMBRANE PROTEIN
Function / homologyOtopetrin / Otopetrin / monoatomic ion transport / membrane => GO:0016020 / plasma membrane / LOC100127796 protein
Function and homology information
Biological speciesXenopus tropicalis (tropical clawed frog)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.92 Å
AuthorsChen QF / Bai XC
Funding support United States, 5 items
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, 2019-
Header (metadata) releaseApr 24, 2019-
Map releaseApr 24, 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: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6o84
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0650.png

Downloads & links

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Map

FileDownload / File: emd_0650.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of OTOP3 from xenopus tropicalis
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.05631465 - 0.11147861
Average (Standard dev.)-0.000003469417 (±0.005685513)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 192.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z192.600192.600192.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.0560.111-0.000

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Supplemental data

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Sample components

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Entire : Xenopus tropicalis OTOP3

EntireName: Xenopus tropicalis OTOP3
Components
  • Organelle or cellular component: Xenopus tropicalis OTOP3
    • Protein or peptide: LOC100127796 protein,LOC100127796 protein,OTOP3,LOC100127796 protein,LOC100127796 protein

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Supramolecule #1: Xenopus tropicalis OTOP3

SupramoleculeName: Xenopus tropicalis OTOP3 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Xenopus tropicalis (tropical clawed frog)
Molecular weightTheoretical: 77 KDa

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Macromolecule #1: LOC100127796 protein,LOC100127796 protein,OTOP3,LOC100127796 prot...

MacromoleculeName: LOC100127796 protein,LOC100127796 protein,OTOP3,LOC100127796 protein,LOC100127796 protein
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus tropicalis (tropical clawed frog)
Molecular weightTheoretical: 73.352297 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MLSKEEPACR QFHSREKTWG NEHNGKTVTQ ANNKEHKVPA VRRGDIGQRE PAAHPASHIQ GTMASENTGE QATCKDQYMD LDAPGNNLE HSWLHRHCEI PTTLHQRAKK TGRLFSGLFG LNLMFLGGTV VSSVALSNKA VPERDSQSFL CILMLLSSVW A LYHLLFIR ...String:
MLSKEEPACR QFHSREKTWG NEHNGKTVTQ ANNKEHKVPA VRRGDIGQRE PAAHPASHIQ GTMASENTGE QATCKDQYMD LDAPGNNLE HSWLHRHCEI PTTLHQRAKK TGRLFSGLFG LNLMFLGGTV VSSVALSNKA VPERDSQSFL CILMLLSSVW A LYHLLFIR NQNGAVHHDH HAGAMWLKAS LAIFGVCSII LSIFEIGHAL LLQNCEILMD IVFFSIEIVF VSVQTVLLWV SC KDCVQMH HSVTRYGIML TLATDILLWL TAVIDDSLEQ DLEILQSNST QDESNEMAQC QCPTDSMCWG LKQGYVTMFP FNI EYSLIC ATLLFIMWKN VGRRE(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)W(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)R KLDVTLLFVS AVGQLGISYF SIIATVVTTP WTML SALNF SNSLLLILQY LSQTMFIIES MRSIHEEEKE KPGHHEESHR RMSVQEMHKA PPSCLDAGHL GLSRRVVKEM AMFLM ICNI MCWILGAFGA HPLYMNGLER QLYGSGIWLA ILNIGLPLSV FYRMHSVGIL LEVYLHALEG SSLVPR

UniProtKB: LOC100127796 protein, LOC100127796 protein

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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

Concentration1.3 mg/mL
BufferpH: 8 / Component - Concentration: 150.0 mM / Component - Formula: NaClSodium chloride / Component - Name: sodium chloride
Details: Solutions were made fresh from stock solutions and filtered.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 46730
Specialist opticsEnergy filter - Name: GIF Quantum LS
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 3000 / Average exposure time: 15.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1433949 / Details: The particles were auto-picked in RELION.
Startup modelType of model: NONE
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 2)
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 2)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2)
Details: Auto-refinement in RELION generated the final reconstruction. The angular sampling was determined automatically in RELION.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.92 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2) / Number images used: 212551
Details30 frames per movie stack were saved for motion correction.

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