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- PDB-5irz: Structure of TRPV1 determined in lipid nanodisc -

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Basic information

Entry
Database: PDB / ID: 5irz
TitleStructure of TRPV1 determined in lipid nanodisc
ComponentsTransient receptor potential cation channel subfamily V member 1
KeywordsTRANSPORT PROTEIN / TRP / ion channel / nanodisc / vanilloid / lipid
Function / homology
Function and homology information


response to capsazepine / negative regulation of establishment of blood-brain barrier / sensory perception of mechanical stimulus / peptide secretion / excitatory extracellular ligand-gated monoatomic ion channel activity / temperature-gated ion channel activity / detection of chemical stimulus involved in sensory perception of pain / cellular response to temperature stimulus / urinary bladder smooth muscle contraction / TRP channels ...response to capsazepine / negative regulation of establishment of blood-brain barrier / sensory perception of mechanical stimulus / peptide secretion / excitatory extracellular ligand-gated monoatomic ion channel activity / temperature-gated ion channel activity / detection of chemical stimulus involved in sensory perception of pain / cellular response to temperature stimulus / urinary bladder smooth muscle contraction / TRP channels / smooth muscle contraction involved in micturition / fever generation / thermoception / detection of temperature stimulus involved in thermoception / cellular response to acidic pH / negative regulation of systemic arterial blood pressure / response to pH / dendritic spine membrane / chloride channel regulator activity / monoatomic cation transmembrane transporter activity / glutamate secretion / negative regulation of heart rate / cellular response to ATP / response to pain / temperature homeostasis / cellular response to alkaloid / diet induced thermogenesis / cellular response to cytokine stimulus / calcium ion import across plasma membrane / intracellularly gated calcium channel activity / behavioral response to pain / detection of temperature stimulus involved in sensory perception of pain / negative regulation of mitochondrial membrane potential / ligand-gated monoatomic ion channel activity / monoatomic cation channel activity / extracellular ligand-gated monoatomic ion channel activity / sensory perception of pain / phosphatidylinositol binding / GABA-ergic synapse / phosphoprotein binding / microglial cell activation / cellular response to nerve growth factor stimulus / cellular response to growth factor stimulus / lipid metabolic process / calcium channel activity / calcium ion transmembrane transport / response to peptide hormone / calcium ion transport / transmembrane signaling receptor activity / positive regulation of nitric oxide biosynthetic process / sensory perception of taste / cellular response to tumor necrosis factor / cellular response to heat / response to heat / positive regulation of cytosolic calcium ion concentration / monoatomic ion transmembrane transport / protein homotetramerization / postsynaptic membrane / calmodulin binding / neuron projection / positive regulation of apoptotic process / external side of plasma membrane / neuronal cell body / dendrite / negative regulation of transcription by RNA polymerase II / ATP binding / identical protein binding / membrane / metal ion binding / plasma membrane
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-6ES / Chem-6O8 / Chem-6OE / Transient receptor potential cation channel subfamily V member 1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsGao, Y. / Cao, E. / Julius, D. / Cheng, Y.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS047723 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R37NS065071 United States
National Institutes of Health/Office of the DirectorS10OD020054 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM098672 United States
CitationJournal: Nature / Year: 2016
Title: TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action.
Authors: Yuan Gao / Erhu Cao / David Julius / Yifan Cheng /
Abstract: When integral membrane proteins are visualized in detergents or other artificial systems, an important layer of information is lost regarding lipid interactions and their effects on protein structure. ...When integral membrane proteins are visualized in detergents or other artificial systems, an important layer of information is lost regarding lipid interactions and their effects on protein structure. This is especially relevant to proteins for which lipids have both structural and regulatory roles. Here we demonstrate the power of combining electron cryo-microscopy with lipid nanodisc technology to ascertain the structure of the rat TRPV1 ion channel in a native bilayer environment. Using this approach, we determined the locations of annular and regulatory lipids and showed that specific phospholipid interactions enhance binding of a spider toxin to TRPV1 through formation of a tripartite complex. Furthermore, phosphatidylinositol lipids occupy the binding site for capsaicin and other vanilloid ligands, suggesting a mechanism whereby chemical or thermal stimuli elicit channel activation by promoting the release of bioactive lipids from a critical allosteric regulatory site.
History
DepositionMar 15, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 25, 2016Provider: repository / Type: Initial release
Revision 1.1Jun 22, 2016Group: Database references
Revision 1.2Nov 30, 2016Group: Source and taxonomy
Revision 1.3Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.4Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Mar 6, 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
D: Transient receptor potential cation channel subfamily V member 1
E: Transient receptor potential cation channel subfamily V member 1
B: Transient receptor potential cation channel subfamily V member 1
C: Transient receptor potential cation channel subfamily V member 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)302,47228
Polymers291,8374
Non-polymers10,63524
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area19020 Å2
ΔGint-205 kcal/mol
Surface area73650 Å2

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Components

#1: Protein
Transient receptor potential cation channel subfamily V member 1 / TrpV1 / Capsaicin receptor / Osm-9-like TRP channel 1 / OTRPC1 / Vanilloid receptor 1 / Vanilloid ...TrpV1 / Capsaicin receptor / Osm-9-like TRP channel 1 / OTRPC1 / Vanilloid receptor 1 / Vanilloid receptor type 1-like


Mass: 72959.297 Da / Num. of mol.: 4 / Fragment: UNP residues 110-603, 627-764
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Trpv1, Vr1, Vr1l / Cell line (production host): HEK 293S / Production host: Homo sapiens (human) / References: UniProt: O35433
#2: Chemical
ChemComp-6O8 / (4R,7S)-4-hydroxy-N,N,N-trimethyl-4,9-dioxo-7-[(pentanoyloxy)methyl]-3,5,8-trioxa-4lambda~5~-phosphatetradecan-1-aminium


Mass: 440.489 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C19H39NO8P
#3: Chemical
ChemComp-6ES / (2S)-1-{[(R)-hydroxy{[(1R,2R,3S,4S,5S,6S)-2,3,4,5,6-pentahydroxycyclohexyl]oxy}phosphoryl]oxy}-3-(pentanoyloxy)propan-2-yl decanoate


Mass: 572.580 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C24H45O13P
#4: Chemical
ChemComp-6OE / (2S)-3-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-2-(hexanoyloxy)propyl hexanoate


Mass: 411.428 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C17H34NO8P

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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: TRPV1 ion channel in unliganded state / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human) / Plasmid: unknown
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
32 mMTCEPC9H15O6P1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K
Details: Blot for 6 seconds before plunging into liquid ethane (FEI VITROBOT MARK III).

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300 / Details: Grid screening was performed manually.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 31000 X / Calibrated magnification: 41132 X / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm / Calibrated defocus min: 700 nm / Calibrated defocus max: 2200 nm / Cs: 2 mm / C2 aperture diameter: 30 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 6 sec. / Electron dose: 41 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1000
Details: Images were collected in movie mode at 5 frames per second for 6 seconds.
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 30 / Used frames/image: 1-30

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Processing

SoftwareName: PHENIX / Version: 1.10_2152: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1SPIDERparticle selectionoperations CA S, CL KM, AP SH
2UCSFImage4image acquisitiondata was manually collected
4CTFFIND44CTF correction
7UCSF Chimera1.1model fittingfit in map
9RELION1.3initial Euler assignment
10RELION1.3final Euler assignment
11RELION1.3classification
12RELION1.33D reconstruction
13Cootmodel refinementmanual adjustment
14PHENIXmodel refinementphenix.real_sapce_refine
Image processingDetails: Each image stack was subjected to whole-frame motion correction followed by correction at the individual pixel level using program UcsfDfCorr.
CTF correctionDetails: CTF correction was performed before classification and refinement.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 159193
Details: Initial manual particle picking and automatic particle picking were performed using SamViewer and several python scripts based on SPIDER.
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30689 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: correlation coefficient
Atomic model buildingPDB-ID: 3J5P

3j5p


Pdb chain-ID: A / Accession code: 3J5P / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.28 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00812076
ELECTRON MICROSCOPYf_angle_d0.91316400
ELECTRON MICROSCOPYf_dihedral_angle_d9.8379280
ELECTRON MICROSCOPYf_chiral_restr0.0561904
ELECTRON MICROSCOPYf_plane_restr0.0052020

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