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- PDB-8gka: Human TRPV3 tetramer structure, closed conformation -

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Basic information

Entry
Database: PDB / ID: 8gka
TitleHuman TRPV3 tetramer structure, closed conformation
ComponentsTransient receptor potential cation channel subfamily V member 3
KeywordsMEMBRANE PROTEIN / Ion Channel / TRP Channel / Closed Conformation
Function / homology
Function and homology information


negative regulation of hair cycle / TRP channels / response to temperature stimulus / positive regulation of calcium ion import / calcium ion transmembrane transport / calcium channel activity / lysosome / receptor complex / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
Transient receptor potential cation channel subfamily V member 1-4 / Transient receptor potential cation channel subfamily V / Ankyrin repeat / 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
Chem-POV / Transient receptor potential cation channel subfamily V member 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.55 Å
AuthorsLansky, S. / Betancourt, J.M. / Scheuring, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) United States
CitationJournal: Nature / Year: 2023
Title: A pentameric TRPV3 channel with a dilated pore.
Authors: Shifra Lansky / John Michael Betancourt / Jingying Zhang / Yining Jiang / Elizabeth D Kim / Navid Paknejad / Crina M Nimigean / Peng Yuan / Simon Scheuring /
Abstract: Transient receptor potential (TRP) channels are a large, eukaryotic ion channel superfamily that control diverse physiological functions, and therefore are attractive drug targets. More than 210 ...Transient receptor potential (TRP) channels are a large, eukaryotic ion channel superfamily that control diverse physiological functions, and therefore are attractive drug targets. More than 210 structures from more than 20 different TRP channels have been determined, and all are tetramers. Despite this wealth of structures, many aspects concerning TRPV channels remain poorly understood, including the pore-dilation phenomenon, whereby prolonged activation leads to increased conductance, permeability to large ions and loss of rectification. Here, we used high-speed atomic force microscopy (HS-AFM) to analyse membrane-embedded TRPV3 at the single-molecule level and discovered a pentameric state. HS-AFM dynamic imaging revealed transience and reversibility of the pentamer in dynamic equilibrium with the canonical tetramer through membrane diffusive protomer exchange. The pentamer population increased upon diphenylboronic anhydride (DPBA) addition, an agonist that has been shown to induce TRPV3 pore dilation. On the basis of these findings, we designed a protein production and data analysis pipeline that resulted in a cryogenic-electron microscopy structure of the TRPV3 pentamer, showing an enlarged pore compared to the tetramer. The slow kinetics to enter and exit the pentameric state, the increased pentamer formation upon DPBA addition and the enlarged pore indicate that the pentamer represents the structural correlate of pore dilation. We thus show membrane diffusive protomer exchange as an additional mechanism for structural changes and conformational variability. Overall, we provide structural evidence for a non-canonical pentameric TRP-channel assembly, laying the foundation for new directions in TRP channel research.
History
DepositionMar 17, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 6, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Sep 20, 2023Group: Database references / Refinement description / Category: citation / em_3d_fitting_list
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_3d_fitting_list.initial_refinement_model_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transient receptor potential cation channel subfamily V member 3
B: Transient receptor potential cation channel subfamily V member 3
C: Transient receptor potential cation channel subfamily V member 3
D: Transient receptor potential cation channel subfamily V member 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)388,07738
Polymers362,9714
Non-polymers25,10534
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, The tetrameric assembly is supported by high-speed atomic force microscopy (HS-AFM) imaging
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Transient receptor potential cation channel subfamily V member 3 / TrpV3 / Vanilloid receptor-like 3 / VRL-3


Mass: 90742.812 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TRPV3 / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q8NET8
#2: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#3: Chemical...
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 33 / Source method: obtained synthetically / Formula: C42H82NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
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: TRPV3 ion channel- tetrameric oligomerization assembly
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293S GnTI-
Buffer solutionpH: 8
Details: 20 mM Tris pH 8, 150 mM NaCl, 0.01% GDN, 1 mM beta-mercaptoethanol
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2150 mMsodium chlorideNaCl1
30.01 %glyco-diosgeninC56H92O251
41 mMbeta-mercaptoethanolC2H6OS1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 3 ul sample blot 2 sec force 3 wait 20 sec

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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 magnification: 64000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 0.001 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 53.27 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 10125

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameCategory
1cryoSPARCparticle selection
2Leginonimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3668517
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.55 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1532979 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 6UW4

6uw4


Accession code: 6uw4 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00122226
ELECTRON MICROSCOPYf_angle_d0.36229826
ELECTRON MICROSCOPYf_dihedral_angle_d12.0898599
ELECTRON MICROSCOPYf_chiral_restr0.0323233
ELECTRON MICROSCOPYf_plane_restr0.0023600

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