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- PDB-5irx: Structure of TRPV1 in complex with DkTx and RTX, determined in li... -

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Basic information

Entry
Database: PDB / ID: 5irx
TitleStructure of TRPV1 in complex with DkTx and RTX, determined in lipid nanodisc
Components
  • Tau-theraphotoxin-Hs1a
  • Transient receptor potential cation channel subfamily V member 1
KeywordsTRANSPORT PROTEIN / TRP / ion channel / nanodisc / vanilloid / lipid
Function / homology
Function and homology information


temperature-gated ion channel activity / response to capsazepine / negative regulation of establishment of blood-brain barrier / sensory perception of mechanical stimulus / peptide secretion / urinary bladder smooth muscle contraction / detection of chemical stimulus involved in sensory perception of pain / smooth muscle contraction involved in micturition / TRP channels / cellular response to temperature stimulus ...temperature-gated ion channel activity / response to capsazepine / negative regulation of establishment of blood-brain barrier / sensory perception of mechanical stimulus / peptide secretion / urinary bladder smooth muscle contraction / detection of chemical stimulus involved in sensory perception of pain / smooth muscle contraction involved in micturition / TRP channels / cellular response to temperature stimulus / cellular response to acidic pH / fever generation / detection of temperature stimulus involved in thermoception / thermoception / ion channel regulator activity / negative regulation of systemic arterial blood pressure / glutamate secretion / chloride channel regulator activity / response to pH / dendritic spine membrane / monoatomic cation transmembrane transporter activity / excitatory extracellular ligand-gated monoatomic ion channel activity / negative regulation of heart rate / cellular response to ATP / temperature homeostasis / response to pain / cellular response to alkaloid / calcium ion import across plasma membrane / behavioral response to pain / diet induced thermogenesis / cellular response to cytokine stimulus / detection of temperature stimulus involved in sensory perception of pain / intracellularly gated calcium channel activity / negative regulation of mitochondrial membrane potential / ligand-gated monoatomic ion channel activity / extracellular ligand-gated monoatomic ion channel activity / monoatomic cation channel activity / GABA-ergic synapse / sensory perception of pain / phosphatidylinositol binding / cellular response to nerve growth factor stimulus / phosphoprotein binding / calcium ion transmembrane transport / microglial cell activation / calcium channel activity / lipid metabolic process / cellular response to growth factor stimulus / response to peptide hormone / calcium ion transport / positive regulation of nitric oxide biosynthetic process / transmembrane signaling receptor activity / cellular response to tumor necrosis factor / cellular response to heat / positive regulation of cytosolic calcium ion concentration / response to heat / monoatomic ion transmembrane transport / toxin activity / postsynaptic membrane / protein homotetramerization / calmodulin binding / neuron projection / positive regulation of apoptotic process / external side of plasma membrane / neuronal cell body / lipid binding / dendrite / negative regulation of transcription by RNA polymerase II / extracellular region / 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
resiniferatoxin / Chem-6O8 / Chem-6O9 / Chem-6OE / Transient receptor potential cation channel subfamily V member 1 / Tau-theraphotoxin-Hs1a
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Haplopelma schmidti (Chinese earth tiger)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.95 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 14, 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

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: 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
D: Transient receptor potential cation channel subfamily V member 1
E: Tau-theraphotoxin-Hs1a
F: Tau-theraphotoxin-Hs1a
hetero molecules


Theoretical massNumber of molelcules
Total (without water)317,87726
Polymers308,9436
Non-polymers8,93320
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21910 Å2
ΔGint-234 kcal/mol
Surface area81540 Å2

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Components

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Protein , 2 types, 6 molecules ABCDEF

#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): HEK293S / Production host: Homo sapiens (human) / References: UniProt: O35433
#2: Protein Tau-theraphotoxin-Hs1a / Tau-TRTX-Hs1a / Double-knot toxin / DkTx


Mass: 8552.988 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Haplopelma schmidti (Chinese earth tiger)
Plasmid: pET19 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0CH43

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Non-polymers , 4 types, 20 molecules

#3: 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
#4: Chemical
ChemComp-6OE / (2S)-3-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-2-(hexanoyloxy)propyl hexanoate


Mass: 411.428 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C17H34NO8P
#5: Chemical
ChemComp-6EU / resiniferatoxin / RTX


Mass: 628.708 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C37H40O9 / Comment: toxin*YM
#6: Chemical
ChemComp-6O9 / (2S)-2-(acetyloxy)-3-{[(R)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}propyl pentanoate


Mass: 341.295 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C12H24NO8P

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1TRPV1 ion channel in complex with DkTx and RTXCOMPLEXTwo molecules of DkTx and 4 molecules of RTX bound to one TRPV1 homo-tetramer#1-#20MULTIPLE SOURCES
2Transient receptor potential cation channel subfamily V member 1COMPLEX#11RECOMBINANT
3Tau-theraphotoxin-Hs1aCOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Rattus norvegicus (Norway rat)10116
23Haplopelma schmidti (Chinese earth tiger)29017
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
12Homo sapiens (human)9606unknown
23Escherichia coli BL21(DE3) (bacteria)469008unknown
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: 1200
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: 218787
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: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73929 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: correlation coefficient
Atomic model buildingPDB-ID: 3J5Q

3j5q


Pdb chain-ID: A
RefinementHighest resolution: 2.95 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01312886
ELECTRON MICROSCOPYf_angle_d1.14717536
ELECTRON MICROSCOPYf_dihedral_angle_d10.4849576
ELECTRON MICROSCOPYf_chiral_restr0.0581990
ELECTRON MICROSCOPYf_plane_restr0.0062172

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