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- PDB-8ddq: cryo-EM structure of TRPM3 ion channel in the presence of soluble... -

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Basic information

Entry
Database: PDB / ID: 8ddq
Titlecryo-EM structure of TRPM3 ion channel in the presence of soluble Gbg, focused on channel
Components
  • Transient receptor potential cation channel, subfamily M, member 3
  • Unidentified segment at the N-terminus of TRPM3
KeywordsMEMBRANE PROTEIN / TRPM3 / ion channel / Gbg
Function / homology
Function and homology information


metal ion transport / monoatomic cation transmembrane transport / monoatomic cation transport / monoatomic cation channel activity / protein tetramerization / plasma membrane
Similarity search - Function
TRPM, tetramerisation domain / TRPM, tetramerisation domain superfamily / Tetramerisation domain of TRPM / TRPM, SLOG domain / : / SLOG in TRPM / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
1,2-DIACYL-GLYCEROL-3-SN-PHOSPHATE / Transient receptor potential cation channel, subfamily M, member 3
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsZhao, C. / MacKinnon, R.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Neuron / Year: 2023
Title: Structural and functional analyses of a GPCR-inhibited ion channel TRPM3.
Authors: Chen Zhao / Roderick MacKinnon /
Abstract: G-protein coupled receptors (GPCRs) govern the physiological response to stimuli by modulating the activity of downstream effectors, including ion channels. TRPM3 is an ion channel inhibited by GPCRs ...G-protein coupled receptors (GPCRs) govern the physiological response to stimuli by modulating the activity of downstream effectors, including ion channels. TRPM3 is an ion channel inhibited by GPCRs through direct interaction with G protein (Gβγ) released upon their activation. This GPCR-TRPM3 signaling pathway contributes to the analgesic effect of morphine. Here, we characterized Gβγ inhibition of TRPM3 using electrophysiology and single particle cryo-electron microscopy (cryo-EM). From electrophysiology, we obtained a half inhibition constant (IC50) of ∼240 nM. Using cryo-EM, we determined structures of mouse TRPM3 expressed in human cells with and without Gβγ and with and without PIP, a lipid required for TRPM3 activity, at resolutions of 2.7-4.7 Å. Gβγ-TRPM3 interfaces vary depending on PIP occupancy; however, in all cases, Gβγ appears loosely attached to TRPM3. The IC50 in electrophysiology experiments raises the possibility that additional unknown factors may stabilize the TRPM3-Gβγ complex.
History
DepositionJun 18, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 2, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 9, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 18, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year
Revision 1.3Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Transient receptor potential cation channel, subfamily M, member 3
E: Unidentified segment at the N-terminus of TRPM3
B: Transient receptor potential cation channel, subfamily M, member 3
F: Unidentified segment at the N-terminus of TRPM3
C: Transient receptor potential cation channel, subfamily M, member 3
G: Unidentified segment at the N-terminus of TRPM3
D: Transient receptor potential cation channel, subfamily M, member 3
H: Unidentified segment at the N-terminus of TRPM3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)630,02618
Polymers624,9818
Non-polymers5,04510
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Transient receptor potential cation channel, subfamily M, member 3


Mass: 154780.516 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Trpm3 / Production host: Homo sapiens (human) / References: UniProt: Q5F4S7
#2: Protein/peptide
Unidentified segment at the N-terminus of TRPM3


Mass: 1464.797 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: a segment at the N-terminus of TRPM3 whose sequence cannot be identified from the cryo-EM density
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#3: Chemical
ChemComp-3PH / 1,2-DIACYL-GLYCEROL-3-SN-PHOSPHATE / PHOSPHATIDIC ACID


Mass: 704.998 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C39H77O8P
#4: Chemical
ChemComp-9Z9 / (3beta,14beta,17beta,25R)-3-[4-methoxy-3-(methoxymethyl)butoxy]spirost-5-en


Mass: 544.805 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H56O5 / Comment: detergent*YM
#5: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
Has ligand of interestN

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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: TRPM3 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 1.35 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1948680 / Symmetry type: POINT
RefinementCross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00332840
ELECTRON MICROSCOPYf_angle_d0.53144424
ELECTRON MICROSCOPYf_dihedral_angle_d11.24712168
ELECTRON MICROSCOPYf_chiral_restr0.0384956
ELECTRON MICROSCOPYf_plane_restr0.0045532

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