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- PDB-8gvx: Cryo-EM structure of the human TRPC5 ion channel in complex with ... -

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Basic information

Entry
Database: PDB / ID: 8gvx
TitleCryo-EM structure of the human TRPC5 ion channel in complex with G alpha i3 subunits, class2
Components
  • Guanine nucleotide-binding protein G(i) subunit alpha-3
  • Short transient receptor potential channel 5
KeywordsMETAL TRANSPORT / TRP / transient receptor potential
Function / homology
Function and homology information


regulation of membrane hyperpolarization / phosphatidylserine exposure on apoptotic cell surface / negative regulation of dendrite morphogenesis / Role of second messengers in netrin-1 signaling / store-operated calcium channel activity / cation channel complex / negative regulation of adenylate cyclase activity / inositol 1,4,5 trisphosphate binding / actinin binding / TRP channels ...regulation of membrane hyperpolarization / phosphatidylserine exposure on apoptotic cell surface / negative regulation of dendrite morphogenesis / Role of second messengers in netrin-1 signaling / store-operated calcium channel activity / cation channel complex / negative regulation of adenylate cyclase activity / inositol 1,4,5 trisphosphate binding / actinin binding / TRP channels / GTP metabolic process / dopamine receptor signaling pathway / clathrin binding / positive regulation of macroautophagy / Adenylate cyclase inhibitory pathway / positive regulation of axon extension / calcium channel complex / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / regulation of cytosolic calcium ion concentration / positive regulation of neuron differentiation / positive regulation of peptidyl-threonine phosphorylation / G protein-coupled receptor binding / calcium ion transmembrane transport / G-protein beta/gamma-subunit complex binding / calcium channel activity / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / ADP signalling through P2Y purinoceptor 12 / neuron differentiation / G alpha (z) signalling events / ADORA2B mediated anti-inflammatory cytokines production / GPER1 signaling / GDP binding / heterotrimeric G-protein complex / calcium ion transport / nervous system development / actin binding / midbody / ATPase binding / positive regulation of cytosolic calcium ion concentration / G alpha (i) signalling events / growth cone / G alpha (s) signalling events / neuron apoptotic process / Extra-nuclear estrogen signaling / cell cycle / cell division / lysosomal membrane / GTPase activity / centrosome / dendrite / neuronal cell body / positive regulation of cell population proliferation / GTP binding / nucleolus / Golgi apparatus / extracellular exosome / nucleoplasm / membrane / metal ion binding / plasma membrane / cytoplasm
Similarity search - Function
Transient receptor potential channel, canonical 5 / Transient receptor ion channel II / Transient receptor ion channel domain / Transient receptor ion channel II / Transient receptor potential channel, canonical / G-protein alpha subunit, group I / Ankyrin repeat / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion ...Transient receptor potential channel, canonical 5 / Transient receptor ion channel II / Transient receptor ion channel domain / Transient receptor ion channel II / Transient receptor potential channel, canonical / G-protein alpha subunit, group I / Ankyrin repeat / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / Ankyrin repeats (3 copies) / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / Ion transport domain / Ion transport protein / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / PHOSPHATIDYLETHANOLAMINE / CHOLESTEROL HEMISUCCINATE / Chem-YZY / Guanine nucleotide-binding protein G(i) subunit alpha-3 / Short transient receptor potential channel 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.91 Å
AuthorsWon, J. / Jeong, H. / Lee, H.H.
Funding support Korea, Republic Of, 1items
OrganizationGrant numberCountry
Other privateSSTF-BA2101-13 Korea, Republic Of
CitationJournal: Nat Commun / Year: 2023
Title: Molecular architecture of the Gα-bound TRPC5 ion channel.
Authors: Jongdae Won / Jinsung Kim / Hyeongseop Jeong / Jinhyeong Kim / Shasha Feng / Byeongseok Jeong / Misun Kwak / Juyeon Ko / Wonpil Im / Insuk So / Hyung Ho Lee /
Abstract: G-protein coupled receptors (GPCRs) and ion channels serve as key molecular switches through which extracellular stimuli are transformed into intracellular effects, and it has long been postulated ...G-protein coupled receptors (GPCRs) and ion channels serve as key molecular switches through which extracellular stimuli are transformed into intracellular effects, and it has long been postulated that ion channels are direct effector molecules of the alpha subunit of G-proteins (Gα). However, no complete structural evidence supporting the direct interaction between Gα and ion channels is available. Here, we present the cryo-electron microscopy structures of the human transient receptor potential canonical 5 (TRPC5)-Gα complexes with a 4:4 stoichiometry in lipid nanodiscs. Remarkably, Gα binds to the ankyrin repeat edge of TRPC5 ~ 50 Å away from the cell membrane. Electrophysiological analysis shows that Gα increases the sensitivity of TRPC5 to phosphatidylinositol 4,5-bisphosphate (PIP), thereby rendering TRPC5 more easily opened in the cell membrane, where the concentration of PIP is physiologically regulated. Our results demonstrate that ion channels are one of the direct effector molecules of Gα proteins triggered by GPCR activation-providing a structural framework for unraveling the crosstalk between two major classes of transmembrane proteins: GPCRs and ion channels.
History
DepositionSep 16, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 24, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Short transient receptor potential channel 5
F: Guanine nucleotide-binding protein G(i) subunit alpha-3
E: Guanine nucleotide-binding protein G(i) subunit alpha-3
G: Guanine nucleotide-binding protein G(i) subunit alpha-3
H: Guanine nucleotide-binding protein G(i) subunit alpha-3
A: Short transient receptor potential channel 5
C: Short transient receptor potential channel 5
D: Short transient receptor potential channel 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)535,18132
Polymers525,4048
Non-polymers9,77824
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 8 molecules BACDFEGH

#1: Protein
Short transient receptor potential channel 5 / TrpC5 / Transient receptor protein 5 / TRP-5 / hTRP-5 / hTRP5


Mass: 89951.891 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: residues 766,767(SR) restriction enzyme, XbaI, residues 768-773(LEVLFQ) protease cleavage site, HRV-3C
Source: (gene. exp.) Homo sapiens (human) / Gene: TRPC5, TRP5 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q9UL62
#2: Protein
Guanine nucleotide-binding protein G(i) subunit alpha-3 / G(i) alpha-3


Mass: 41399.047 Da / Num. of mol.: 4 / Mutation: Q204L
Source method: isolated from a genetically manipulated source
Details: 6 histidine tag is inserted between M119 and T120. / Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI3 / Production host: Escherichia coli K-12 (bacteria) / Strain (production host): K-12 / References: UniProt: P08754

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Non-polymers , 6 types, 24 molecules

#3: Chemical
ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE / Phosphatidylethanolamine


Mass: 734.039 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C40H80NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#4: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C31H50O4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical
ChemComp-YZY / (2S)-2-(hexadecanoyloxy)-3-hydroxypropyl (9Z)-octadec-9-enoate


Mass: 594.949 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C37H70O5 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM

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Details

Has ligand of interestY

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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: Transient receptor potentialTransient receptor potential channel
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.91 Å / Resolution method: OTHER / Num. of particles: 5344
Details: We combined two different maps from the same dataset (D_1300031718_em-additional-volume_P1.map.V4 and D_1300031718_em-additional-volume_P2.map.V4) to generate a composite map (D_1300031718_ ...Details: We combined two different maps from the same dataset (D_1300031718_em-additional-volume_P1.map.V4 and D_1300031718_em-additional-volume_P2.map.V4) to generate a composite map (D_1300031718_em-volume_P1.map.V6). The density of the G protein area could not be visualized clearly in the consensus map of this EM dataset. Therefore, we performed focused classification and local refinement to improve the density of the G protein area using symmetry expanded particles with C4 symmetry imposition, which required more number of particles. Finally, the number of particles used to reconstruct additional volume data 1 (D_1300031718_em-additional-volume_P1.map.V4) is 5,344 and the number of particles used to reconstruct additional volume data 2 (D_1300031718_em-additional-volume_P2.map.V4) is 205,343. Furthermore, the resolution stated above is based on map resolution estimates calculated by a validation tool in Phenix, FSC (model) = 0.143.
Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00433164
ELECTRON MICROSCOPYf_angle_d0.79144864
ELECTRON MICROSCOPYf_dihedral_angle_d6.1054720
ELECTRON MICROSCOPYf_chiral_restr0.0415040
ELECTRON MICROSCOPYf_plane_restr0.0065580

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