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- EMDB-8117: Structure of TRPV1 in complex with DkTx and RTX, determined in li... -

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Basic information

Entry
Database: EMDB / ID: EMD-8117
TitleStructure of TRPV1 in complex with DkTx and RTX, determined in lipid nanodisc
Map dataTRPV1 in complex with DkTx and RTX
Sample
  • Complex: TRPV1 ion channel in complex with DkTx and RTX
    • Complex: Transient receptor potential cation channel subfamily V member 1
      • Protein or peptide: Transient receptor potential cation channel subfamily V member 1
    • Complex: Tau-theraphotoxin-Hs1a
      • Protein or peptide: Tau-theraphotoxin-Hs1a
  • Ligand: (4R,7S)-4-hydroxy-N,N,N-trimethyl-4,9-dioxo-7-[(pentanoyloxy)methyl]-3,5,8-trioxa-4lambda~5~-phosphatetradecan-1-aminium
  • Ligand: (2S)-3-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-2-(hexanoyloxy)propyl hexanoate
  • Ligand: resiniferatoxin
  • Ligand: (2S)-2-(acetyloxy)-3-{[(R)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}propyl pentanoate
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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsGao Y / Cao E / Julius D / Cheng Y
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS047723 United States
National Institutes of Health/Office of the DirectorS10OD020054 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R37NS065071 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
DepositionMay 16, 2016-
Header (metadata) releaseMay 25, 2016-
Map releaseMay 25, 2016-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5irx
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8117.png

Downloads & links

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Map

FileDownload / File: emd_8117.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTRPV1 in complex with DkTx and RTX
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.22 Å/pix.
x 192 pix.
= 233.395 Å		1.22 Å/pix.
x 192 pix.
= 233.395 Å		1.22 Å/pix.
x 192 pix.
= 233.395 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2156 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 3.5 / Movie #1: 4
Minimum - Maximum-14.310628 - 24.29218
Average (Standard dev.)-0.0000000041 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 233.3952 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21559895833331.21559895833331.2155989583333
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z233.395233.395233.395
α/β/γ90.00090.00090.000
start NX/NY/NZ-25-77-48
NX/NY/NZ899896
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-14.31124.292-0.000

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Supplemental data

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Half map: TRPV1 in complex with DkTx and RTX, half map 1

Fileemd_8117_half_map_1.map
AnnotationTRPV1 in complex with DkTx and RTX, half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: TRPV1 in complex with DkTx and RTX, half map 2

Fileemd_8117_half_map_2.map
AnnotationTRPV1 in complex with DkTx and RTX, half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TRPV1 ion channel in complex with DkTx and RTX

EntireName: TRPV1 ion channel in complex with DkTx and RTX
Components
  • Complex: TRPV1 ion channel in complex with DkTx and RTX
    • Complex: Transient receptor potential cation channel subfamily V member 1
      • Protein or peptide: Transient receptor potential cation channel subfamily V member 1
    • Complex: Tau-theraphotoxin-Hs1a
      • Protein or peptide: Tau-theraphotoxin-Hs1a
  • Ligand: (4R,7S)-4-hydroxy-N,N,N-trimethyl-4,9-dioxo-7-[(pentanoyloxy)methyl]-3,5,8-trioxa-4lambda~5~-phosphatetradecan-1-aminium
  • Ligand: (2S)-3-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-2-(hexanoyloxy)propyl hexanoate
  • Ligand: resiniferatoxin
  • Ligand: (2S)-2-(acetyloxy)-3-{[(R)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}propyl pentanoate

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Supramolecule #1: TRPV1 ion channel in complex with DkTx and RTX

SupramoleculeName: TRPV1 ion channel in complex with DkTx and RTX / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Two molecules of DkTx and 4 molecules of RTX bound to one TRPV1 homo-tetramer

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Supramolecule #2: Transient receptor potential cation channel subfamily V member 1

SupramoleculeName: Transient receptor potential cation channel subfamily V member 1
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant plasmid: unknown

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Supramolecule #3: Tau-theraphotoxin-Hs1a

SupramoleculeName: Tau-theraphotoxin-Hs1a / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Haplopelma schmidti (Chinese earth tiger)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: unknown

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Macromolecule #1: Transient receptor potential cation channel subfamily V member 1

MacromoleculeName: Transient receptor potential cation channel subfamily V member 1
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 72.959297 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: AMGSRLYDRR SIFDAVAQSN CQELESLLPF LQRSKKRLTD SEFKDPETGK TCLLKAMLNL HNGQNDTIAL LLDVARKTDS LKQFVNASY TDSYYKGQTA LHIAIERRNM TLVTLLVENG ADVQAAANGD FFKKTKGRPG FYFGELPLSL AACTNQLAIV K FLLQNSWQ ...String:
AMGSRLYDRR SIFDAVAQSN CQELESLLPF LQRSKKRLTD SEFKDPETGK TCLLKAMLNL HNGQNDTIAL LLDVARKTDS LKQFVNASY TDSYYKGQTA LHIAIERRNM TLVTLLVENG ADVQAAANGD FFKKTKGRPG FYFGELPLSL AACTNQLAIV K FLLQNSWQ PADISARDSV GNTVLHALVE VADNTVDNTK FVTSMYNEIL ILGAKLHPTL KLEEITNRKG LTPLALAASS GK IGVLAYI LQREIHEPEC RHLSRKFTEW AYGPVHSSLY DLSCIDTCEK NSVLEVIAYS SSETPNRHDM LLVEPLNRLL QDK WDRFVK RIFYFNFFVY CLYMIIFTAA AYYRPVEGLP PYKLKNTVGD YFRVTGEILS VSGGVYFFFR GIQYFLQRRP SLKS LFVDS YSEILFFVQS LFMLVSVVLY FSQRKEYVAS MVFSLAMGWT NMLYYTRGFQ QMGIYAVMIE KMILRDLCRF MFVYL VFLF GFSTAVVTLI EDGKYNSLYS TCLELFKFTI GMGDLEFTEN YDFKAVFIIL LLAYVILTYI LLLNMLIALM GETVNK IAQ ESKNIWKLQR AITILDTEKS FLKCMRKAFR SGKLLQVGFT PDGKDDYRWC FRVDEVNWTT WNTNVGIINE DPG

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Macromolecule #2: Tau-theraphotoxin-Hs1a

MacromoleculeName: Tau-theraphotoxin-Hs1a / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Haplopelma schmidti (Chinese earth tiger)
Molecular weightTheoretical: 8.552988 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
DCAKEGEVCS WGKKCCDLDN FYCPMEFIPH CKKYKPYVPV TTNCAKEGEV CGWGSKCCHG LDCPLAFIPY CEKYR

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Macromolecule #3: (4R,7S)-4-hydroxy-N,N,N-trimethyl-4,9-dioxo-7-[(pentanoyloxy)meth...

MacromoleculeName: (4R,7S)-4-hydroxy-N,N,N-trimethyl-4,9-dioxo-7-[(pentanoyloxy)methyl]-3,5,8-trioxa-4lambda~5~-phosphatetradecan-1-aminium
type: ligand / ID: 3 / Number of copies: 4 / Formula: 6O8
Molecular weightTheoretical: 440.489 Da
Chemical component information

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

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Macromolecule #4: (2S)-3-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-2-(hexanoylo...

MacromoleculeName: (2S)-3-{[(S)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}-2-(hexanoyloxy)propyl hexanoate
type: ligand / ID: 4 / Number of copies: 8 / Formula: 6OE
Molecular weightTheoretical: 411.428 Da
Chemical component information

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

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Macromolecule #5: resiniferatoxin

MacromoleculeName: resiniferatoxin / type: ligand / ID: 5 / Number of copies: 4 / Formula: 6EU
Molecular weightTheoretical: 628.708 Da
Chemical component information

ChemComp-6EU:
resiniferatoxin / toxin*YM

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Macromolecule #6: (2S)-2-(acetyloxy)-3-{[(R)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy...

MacromoleculeName: (2S)-2-(acetyloxy)-3-{[(R)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}propyl pentanoate
type: ligand / ID: 6 / Number of copies: 4 / Formula: 6O9
Molecular weightTheoretical: 341.295 Da
Chemical component information

ChemComp-6O9:
(2S)-2-(acetyloxy)-3-{[(R)-(2-aminoethoxy)(hydroxy)phosphoryl]oxy}propyl pentanoate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride
2.0 mMC9H15O6PTCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 290.0 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK III
Details: Blot for 6 seconds before plunging into liquid ethane (FEI VITROBOT MARK III)..
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 70.0 K / Max: 70.0 K
DetailsGrid screening was performed manually.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-30 / Number grids imaged: 1 / Number real images: 1200 / Average exposure time: 6.0 sec. / Average electron dose: 41.0 e/Å2
Details: Images were collected in movie mode at 5 frames per second for 6 seconds.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Calibrated defocus max: 2.2 µm / Calibrated defocus min: 0.7 µm / Calibrated magnification: 41132 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 31000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsEach image stack was subjected to whole-frame motion correction followed by correction at the individual pixel level using program UcsfDfCorr.
Particle selectionNumber selected: 218787
Details: Initial manual particle picking and automatic particle picking were performed using SamViewer and several python scripts based on SPIDER.
CTF correctionSoftware - Name: CTFFIND4 (ver. 4)
Details: CTF correction was performed before classification and refinement.
Startup modelType of model: EMDB MAP
EMDB ID:

5776

Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 73929
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final 3D classificationNumber classes: 6 / Avg.num./class: 40000 / Software - Name: RELION (ver. 1.3)
Details: 3D classification yielded two major classes representing ion channel-like structure features.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3j5q


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient
Output model

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

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