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- EMDB-8120: Structure of TRPV1 in complex with capsazepine, determined in lip... -

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Basic information

Entry
Database: EMDB / ID: EMD-8120
TitleStructure of TRPV1 in complex with capsazepine, determined in lipid nanodisc
Map dataNone
Sample
  • Complex: TRPV1 ion channel in complex with capsazepine
    • Protein or peptide: The capsaicin receptor, TRPV1
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 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
DepositionMar 15, 2016-
Header (metadata) releaseJun 15, 2016-
Map releaseJun 15, 2016-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8120.png

Downloads & links

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Map

FileDownload / File: emd_8120.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.2156 Å
Density
Contour LevelBy EMDB: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.06494258 - 0.12037598
Average (Standard dev.)0.00001140693 (±0.006749262)
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-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0650.1200.000

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Supplemental data

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Half map: None

Fileemd_8120_half_map_1.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: None

Fileemd_8120_half_map_2.map
AnnotationNone
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 capsazepine

EntireName: TRPV1 ion channel in complex with capsazepine
Components
  • Complex: TRPV1 ion channel in complex with capsazepine
    • Protein or peptide: The capsaicin receptor, TRPV1

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Supramolecule #1: TRPV1 ion channel in complex with capsazepine

SupramoleculeName: TRPV1 ion channel in complex with capsazepine / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293S

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Macromolecule #1: The capsaicin receptor, TRPV1

MacromoleculeName: The capsaicin receptor, TRPV1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: AMGSRLYDRR SIFDAVAQSN CQELESLLPF LQRSKKRLTD SEFKDPETGK TCLLKAMLNL HNGQNDTIAL LLDVARKTDS LKQFVNASYT DSYYKGQTAL HIAIERRNMT LVTLLVENGA DVQAAANGDF FKKTKGRPGF YFGELPLSLA ACTNQLAIVK FLLQNSWQPA ...String:
AMGSRLYDRR SIFDAVAQSN CQELESLLPF LQRSKKRLTD SEFKDPETGK TCLLKAMLNL HNGQNDTIAL LLDVARKTDS LKQFVNASYT DSYYKGQTAL HIAIERRNMT LVTLLVENGA DVQAAANGDF FKKTKGRPGF YFGELPLSLA ACTNQLAIVK FLLQNSWQPA DISARDSVGN TVLHALVEVA DNTVDNTKFV TSMYNEILIL GAKLHPTLKL EEITNRKGLT PLALAASSGK IGVLAYILQR EIHEPECRHL SRKFTEWAYG PVHSSLYDLS CIDTCEKNSV LEVIAYSSSE TPNRHDMLLV EPLNRLLQDK WDRFVKRIFY FNFFVYCLYM IIFTAAAYYR PVEGLPPYKL KNTVGDYFRV TGEILSVSGG VYFFFRGIQY FLQRRPSLKS LFVDSYSEIL FFVQSLFMLV SVVLYFSQRK EYVASMVFSL AMGWTNMLYY TRGFQQMGIY AVMIEKMILR DLCRFMFVYL VFLFGFSTAV VTLIEDGKYN SLYSTCLELF KFTIGMGDLE FTENYDFKAV FIILLLAYVI LTYILLLNML IALMGETVNK IAQESKNIWK LQRAITILDT EKSFLKCMRK AFRSGKLLQV GFTPDGKDDY RWCFRVDEVN WTTWNTNVGI INEDPG

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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 chloridesodium 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: 298 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
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 FIELDBright-field microscopy / 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
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: 1002 / 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.
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 81709
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:

5778

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final 3D classificationNumber classes: 6 / Avg.num./class: 15000 / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 47477
DetailsEach image stack was subjected to whole-frame motion correction using MotionCorr.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3j5p


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

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