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- PDB-6oo5: Cryo-EM structure of the C2-symmetric TRPV2/RTx complex in amphip... -

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Basic information

Entry
Database: PDB / ID: 6oo5
TitleCryo-EM structure of the C2-symmetric TRPV2/RTx complex in amphipol resolved to 4.2 A
ComponentsTRPV2
KeywordsMETAL TRANSPORT / ion channel / calcium channel / TRP channel
Function / homology
Function and homology information


growth cone membrane / response to temperature stimulus / positive regulation of calcium ion import / calcium ion import across plasma membrane / axonal growth cone / positive regulation of axon extension / calcium channel activity / positive regulation of cold-induced thermogenesis / cell body / cell surface / identical protein binding
Similarity search - Function
Transient receptor potential cation channel subfamily V member 1-4 / Transient receptor potential cation channel subfamily V / 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
resiniferatoxin / Transient receptor potential cation channel subfamily V member 2
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsZubcevic, L. / Hsu, A.L. / Borgnia, M.J. / Lee, S.-Y.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R35NS097241 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ZIC ES103326 United States
CitationJournal: Elife / Year: 2019
Title: Symmetry transitions during gating of the TRPV2 ion channel in lipid membranes.
Authors: Lejla Zubcevic / Allen L Hsu / Mario J Borgnia / Seok-Yong Lee /
Abstract: The Transient Receptor Potential Vanilloid 2 (TRPV2) channel is a member of the temperature-sensing thermoTRPV family. Recent advances in cryo-electronmicroscopy (cryo-EM) and X-ray crystallography ...The Transient Receptor Potential Vanilloid 2 (TRPV2) channel is a member of the temperature-sensing thermoTRPV family. Recent advances in cryo-electronmicroscopy (cryo-EM) and X-ray crystallography have provided many important insights into the gating mechanisms of thermoTRPV channels. Interestingly, crystallographic studies of ligand-dependent TRPV2 gating have shown that the TRPV2 channel adopts two-fold symmetric arrangements during the gating cycle. However, it was unclear if crystal packing forces played a role in stabilizing the two-fold symmetric arrangement of the channel. Here, we employ cryo-EM to elucidate the structure of full-length rabbit TRPV2 in complex with the agonist resiniferatoxin (RTx) in nanodiscs and amphipol. We show that RTx induces two-fold symmetric conformations of TRPV2 in both environments. However, the two-fold symmetry is more pronounced in the native-like lipid environment of the nanodiscs. Our data offers insights into a gating pathway in TRPV2 involving symmetry transitions.
History
DepositionApr 22, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 29, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: TRPV2
B: TRPV2
C: TRPV2
D: TRPV2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)357,4068
Polymers354,8914
Non-polymers2,5154
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17840 Å2
ΔGint-211 kcal/mol
Surface area117090 Å2

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Components

#1: Protein
TRPV2


Mass: 88722.680 Da / Num. of mol.: 4 / Mutation: F470S, L505M, L508T, Q528E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Gene: TRPV2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: G1SNM3
#2: Chemical
ChemComp-6EU / resiniferatoxin / RTX


Mass: 628.708 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C37H40O9 / Comment: toxin*YM

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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: TRPV2 / Type: COMPLEX
Details: TRPV2 in complex with RTx reconstituted into amphipol A8-35
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.300 MDa / Experimental value: NO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Cell: Sf9
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
250 mMTris1
32 uMresiniferatoxin1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: TRPV2 in complex with RTx reconstituted into amphipol A8-35, monodisperse
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 296 K / Details: Blotted 3 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: OTHER / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 42 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 1293

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Processing

SoftwareName: PHENIX / Version: 1.15_3459: / Classification: refinement
EM software
IDNameVersionCategoryDetails
4RELION3CTF correctionGctf was used to perform CTF correction.
7Coot0.8.2model fittingModel was built into the density in Coot.
9PHENIX1.15model refinementPhenix real space refine was performed.
10RELION3initial Euler assignment
12RELION3classificationClassification was performed in RELION 3.0.
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 90862 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5AN8

5an8


Pdb chain-ID: A / Accession code: 5AN8 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00718004
ELECTRON MICROSCOPYf_angle_d0.94624726
ELECTRON MICROSCOPYf_dihedral_angle_d11.72710276
ELECTRON MICROSCOPYf_chiral_restr0.052930
ELECTRON MICROSCOPYf_plane_restr0.0073072

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