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- EMDB-7637: Cytoplasmic domain of mTRPC6 -

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Basic information

Entry
Database: EMDB / ID: EMD-7637
TitleCytoplasmic domain of mTRPC6
Map dataCytoplasmic domain of mTRPC6
Sample
  • Complex: cytoplasmic domain of TRPC6
    • Protein or peptide: Short transient receptor potential channel 6
KeywordsIon Channel / Membrane Protein / TRP Channel / TRPC6 / focal segmental glomerulosclerosis / TRANSPORT PROTEIN
Function / homology
Function and homology information


Elevation of cytosolic Ca2+ levels / Effects of PIP2 hydrolysis / positive regulation of ion transmembrane transporter activity / manganese ion transport / slit diaphragm / negative regulation of dendrite morphogenesis / : / store-operated calcium channel activity / cation channel complex / TRP channels ...Elevation of cytosolic Ca2+ levels / Effects of PIP2 hydrolysis / positive regulation of ion transmembrane transporter activity / manganese ion transport / slit diaphragm / negative regulation of dendrite morphogenesis / : / store-operated calcium channel activity / cation channel complex / TRP channels / inositol 1,4,5 trisphosphate binding / positive regulation of calcium ion transport / actinin binding / clathrin binding / single fertilization / plasma membrane => GO:0005886 / monoatomic cation channel activity / regulation of cytosolic calcium ion concentration / positive regulation of neuron differentiation / positive regulation of peptidyl-threonine phosphorylation / calcium ion transmembrane transport / calcium channel activity / neuron differentiation / cellular response to hydrogen peroxide / monoatomic ion channel activity / actin binding / ATPase binding / positive regulation of cytosolic calcium ion concentration / cellular response to hypoxia / protein homodimerization activity / membrane / plasma membrane / cytoplasm
Similarity search - Function
Transient receptor potential channel, canonical 6 / Transient receptor ion channel domain / Transient receptor ion channel II / Domain of unknown function DUF3447 / Transient receptor potential channel, canonical / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat ...Transient receptor potential channel, canonical 6 / Transient receptor ion channel domain / Transient receptor ion channel II / Domain of unknown function DUF3447 / Transient receptor potential channel, canonical / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Short transient receptor potential channel 6
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsAzumaya CM / Sierra-Valdez FJ
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)R01HD061543 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM125629 United States
CitationJournal: J Biol Chem / Year: 2018
Title: Cryo-EM structure of the cytoplasmic domain of murine transient receptor potential cation channel subfamily C member 6 (TRPC6).
Authors: Caleigh M Azumaya / Francisco Sierra-Valdez / Julio F Cordero-Morales / Terunaga Nakagawa /
Abstract: The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration ...The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration filter, whose damage leads to progressive kidney failure and focal segmental glomerulosclerosis (FSGS). The canonical transient receptor potential 6 (TRPC6) ion channel is expressed in the podocyte, and mutations in its cytoplasmic domain cause FSGS in humans. evaluation of disease-causing mutations in TRPC6 has revealed that these genetic alterations result in abnormal ion channel gating. However, the mechanism whereby the cytoplasmic domain modulates TRPC6 function is largely unknown. Here, we report a cryo-EM structure of the cytoplasmic domain of murine TRPC6 at 3.8 Å resolution. The cytoplasmic fold of TRPC6 is characterized by an inverted dome-like chamber pierced by four radial horizontal helices that converge into a vertical coiled-coil at the central axis. Unlike other TRP channels, TRPC6 displays a unique domain swap that occurs at the junction of the horizontal helices and coiled-coil. Multiple FSGS mutations converge at the buried interface between the vertical coiled-coil and the ankyrin repeats, which form the dome, suggesting these regions are critical for allosteric gating modulation. This functionally critical interface is a potential target for drug design. Importantly, dysfunction in other family members leads to learning deficits (TRPC1/4/5) and ataxia (TRPC3). Our data provide a structural framework for the mechanistic investigation of the TRPC family.
History
DepositionMar 27, 2018-
Header (metadata) releaseApr 18, 2018-
Map releaseMay 23, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0554
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0554
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6cv9
  • Surface level: 0.0554
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7637.png

Downloads & links

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Map

FileDownload / File: emd_7637.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCytoplasmic domain of mTRPC6
Voxel sizeX=Y=Z: 1.41 Å
Density
Contour LevelBy AUTHOR: 0.0554 / Movie #1: 0.0554
Minimum - Maximum-0.12568067 - 0.27699384
Average (Standard dev.)0.00030024402 (±0.005390558)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 360.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.411.411.41
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z360.960360.960360.960
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ242496
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1260.2770.000

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Supplemental data

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Mask #1

Fileemd_7637_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap1 for cytoplasmic domain of mTRPC6

Fileemd_7637_half_map_1.map
AnnotationHalfmap1 for cytoplasmic domain of mTRPC6
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap2 for cytoplasmic domain of mTRPC6

Fileemd_7637_half_map_2.map
AnnotationHalfmap2 for cytoplasmic domain of mTRPC6
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : cytoplasmic domain of TRPC6

EntireName: cytoplasmic domain of TRPC6
Components
  • Complex: cytoplasmic domain of TRPC6
    • Protein or peptide: Short transient receptor potential channel 6

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Supramolecule #1: cytoplasmic domain of TRPC6

SupramoleculeName: cytoplasmic domain of TRPC6 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 144 KDa

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Macromolecule #1: Short transient receptor potential channel 6

MacromoleculeName: Short transient receptor potential channel 6 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 96.671375 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: AMGSMLSIEE ERFLDAAEYG NIPVVRKMLE ECHSLNVNCV DYMGQNALQL AVANEHLEIT ELLLKKENLS RVGDALLLAI SKGYVRIVE AILNHPAFAE GKRLATSPSQ SELQQDDFYA YDEDGTRFSH DVTPIILAAH CQEYEIVHTL LRKGARIERP H DYFCKCTE ...String:
AMGSMLSIEE ERFLDAAEYG NIPVVRKMLE ECHSLNVNCV DYMGQNALQL AVANEHLEIT ELLLKKENLS RVGDALLLAI SKGYVRIVE AILNHPAFAE GKRLATSPSQ SELQQDDFYA YDEDGTRFSH DVTPIILAAH CQEYEIVHTL LRKGARIERP H DYFCKCTE CSQKQKHDSF SHSRSRINAY KGLASPAYLS LSSEDPVMTA LELSNELAVL ANIEKEFKND YRKLSMQCKD FV VGLLDLC RNTEEVEAIL NGDAETRQPG DFGRPNLSRL KLAIKYEVKK FVAHPNCQQQ LLSIWYENLS GLRQQTMAVK FLV VLAVAI GLPFLALIYW CAPCSKMGKI LRGPFMKFVA HAASFTIFLG LLVMNAADRF EGTKLLPNET STDNARQLFR MKTS CFSWM EMLIISWVIG MIWAECKEIW TQGPKEYLFE LWNMLDFGML AIFAASFIAR FMAFWHASKA QSIIDANDTL KDLTK VTLG DNVKYYNLAR IKWDPTDPQI ISEGLYAIAV VLSFSRIAYI LPANESFGPL QISLGRTVKD IFKFMVIFIM VFVAFM IGM FNLYSYYIGA KQNEAFTTVE ESFKTLFWAI FGLSEVKSVV INYNHKFIEN IGYVLYGVYN VTMVIVLLNM LIAMINS SF QEIEDDADVE WKFARAKLWF SYFEEGRTLP VPFNLVPSPK SLLYLLLKFK KWMCELIQGQ KQGFQEDAEM NKRNEEKK F GISGSHEDLS KFSLDKNQLA HNKQSSTRSS EDYHLNSFSN PPRQYQKIMK RLIKRYVLQA QIDKESDEVN EGELKEIKQ DISSLRYELL EEKSQNTEDL AELIRKLGER LSLEPKLEES RR

UniProtKB: Short transient receptor potential channel 6

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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

Concentration0.3 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
4.0 mMTCEPtris(2-carboxyethyl)phosphine
20.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR / Details: 25 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK III
Details: blotted for 8 seconds, 3 filter papers on each side.
DetailsMonodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.001 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 89000
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs aberration corrector
Energy filter - Name: GIF Bioquantum / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
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: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-30 / Number grids imaged: 2 / Number real images: 2618 / Average exposure time: 9.0 sec. / Average electron dose: 46.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 663034
Startup modelType of model: RANDOM CONICAL TILT / Random conical tilt - Tilt angle: 50 degrees
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 200 / Avg.num./class: 2500 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 67280
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 113 / Target criteria: Correlation coefficient
Output model

PDB-6cv9:
Cytoplasmic domain of mTRPC6

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