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- EMDB-20145: Cryo-EM structure of the C2-symmetric TRPV2/RTx complex in amphip... -

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Basic information

Entry
Database: EMDB / ID: EMD-20145
TitleCryo-EM structure of the C2-symmetric TRPV2/RTx complex in amphipol resolved to 3.3 A
Map dataC2-symmetric TRPV2/RTx complex in amphipol resolved to 3.3 A
Sample
  • Complex: TRPV2
    • Protein or peptide: TRPV2
  • Ligand: resiniferatoxin
Function / homology
Function and homology information


growth cone membrane / response to temperature stimulus / positive regulation of calcium ion import / positive regulation of axon extension / axonal growth cone / calcium channel activity / cell body / positive regulation of cold-induced thermogenesis / 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
Transient receptor potential cation channel subfamily V member 2
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsZubcevic L / Hsu AL / Borgnia MJ / Lee S-Y
Funding support United States, 2 items
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, 2019-
Header (metadata) releaseMay 1, 2019-
Map releaseMay 29, 2019-
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: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6oo4
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20145.png

Downloads & links

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Map

FileDownload / File: emd_20145.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC2-symmetric TRPV2/RTx complex in amphipol resolved to 3.3 A
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.17929801 - 0.31808865
Average (Standard dev.)-0.000033027 (±0.011134098)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z276.480276.480276.480
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1790.318-0.000

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Supplemental data

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Half map: Half map 1

Fileemd_20145_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_20145_half_map_2.map
AnnotationHalf 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 : TRPV2

EntireName: TRPV2
Components
  • Complex: TRPV2
    • Protein or peptide: TRPV2
  • Ligand: resiniferatoxin

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Supramolecule #1: TRPV2

SupramoleculeName: TRPV2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: TRPV2 in complex with RTx reconstituted into amphipol A8-35
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: TRPV2

MacromoleculeName: TRPV2 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 88.72268 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MTSPSSPPAF RLETSDGGQD GAEVDKAQLG YGAGPPPMES RFQDEDRNFP PQIKVNLNYR KGAGASQPDL NRFDRDRLFN VVARGNPED LAGLLEYLRR TSKYLTDSEY TEGSTGKTCL MKAVLNLQDG VNACIQPLLE IDRDSGNPQP LVNAQCTDEY Y RGHSALHI ...String:
MTSPSSPPAF RLETSDGGQD GAEVDKAQLG YGAGPPPMES RFQDEDRNFP PQIKVNLNYR KGAGASQPDL NRFDRDRLFN VVARGNPED LAGLLEYLRR TSKYLTDSEY TEGSTGKTCL MKAVLNLQDG VNACIQPLLE IDRDSGNPQP LVNAQCTDEY Y RGHSALHI AIEKRSLQCV KLLVENGANV HAKACGHFFQ KNQDTCFYFG ELPLSLAACT KQWDVVNYLL ENPHQPASLQ AQ DSLGNTV LHALVMIADD SAENSALVVR MYDGLLQAGA RLCPNVQLEG IPNLEGLTPL KLAAKEGKIE IFKHILQREF SAP CQSLSR KFTEWCYGPV RVSLYDLASV DSWEENSVLE IIAFHSRSPH RHRMVVLEPL NKLLQAKWDR LIPRFCFNFL CYLV YMLIF TAVAYHQPAL EKQGFPPLKA TAGNSMLLLG HILILLGGVY LLLGQLWYFW RRRLFIWISF MDSYSEILFL LQALL TVLS QVLCFLAIEW YLPLLVSSLA MGWTNLLYYT RGFQHTGIYS VMIEKVILRD LLRFLLVYLV FLFGFAVALV SLSREA QNS RTPAGPNATE VGQPGAGQED EAPPYRSILD ASLELFKFTI GMGELAFQEQ LRFRGVVLLL LLAYVLLTYV LLLNMLI AL MSETVNSVAT DSWSIWKLQK AISVLEMENG YWWCRRKKQR AGVMLTVGTR PDGSPDERWC FRVGEMNWAT WEQTLPRT L CEEPSGAAAP GVMKNPTPAS QRGEDSASEE DHLPLQLLQS RSNLEVLFQG PHHHHHHDYK DDDDK

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Macromolecule #2: resiniferatoxin

MacromoleculeName: resiniferatoxin / type: ligand / ID: 2 / Number of copies: 4 / Formula: 6EU
Molecular weightTheoretical: 628.708 Da
Chemical component information

ChemComp-6EU:
resiniferatoxin / toxin*YM / Resiniferatoxin

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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

Concentration2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
50.0 mMTris
2.0 uMresiniferatoxin
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 296 K / Instrument: LEICA EM GP / Details: Blotted 3 seconds before plunging.
DetailsTRPV2 in complex with RTx reconstituted into amphipol A8-35, monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Number real images: 1293 / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: RELION (ver. 3.0)
Software - details: Gctf was used to perform CTF correction.
Startup modelType of model: OTHER
Details: An initial model was generated from the 2D particles using the Stochastic Gradient Descent (SGD) algorithm as implemented in RELION 3.0.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 3.0)
Software - details: Classification was performed in RELION 3.0.
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 109623
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5an8


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6oo4:
Cryo-EM structure of the C2-symmetric TRPV2/RTx complex in amphipol resolved to 3.3 A

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