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- EMDB-6267: Human TRPA1 ion channel with agonist AITC -

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Basic information

Entry
Database: EMDB / ID: 6267
TitleHuman TRPA1 ion channel with agonist AITC
Map dataReconstruction of hTRPA1 treated with AITC comprising summed half-maps, unfiltered and unsharpened. Two associated maps are low-pass-filtered and negative-B-factor-sharpened.
SampleRecombinant human TRPA1 treated with AITC:
TRPA1
KeywordsTRPA1 / TRP / ion channel
Function / homologyAnkyrin repeat-containing domain / Ion transport protein / Transient receptor potential cation channel subfamily A member 1 / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Bacterial extracellular solute-binding protein / Ion transport domain / Ankyrin repeat-containing domain superfamily / Ankyrin repeats (3 copies) / Solute-binding family 1, conserved site / Ankyrin repeat ...Ankyrin repeat-containing domain / Ion transport protein / Transient receptor potential cation channel subfamily A member 1 / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Bacterial extracellular solute-binding protein / Ion transport domain / Ankyrin repeat-containing domain superfamily / Ankyrin repeats (3 copies) / Solute-binding family 1, conserved site / Ankyrin repeat / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding proteins, family 1 signature. / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / TRP channels / temperature-gated cation channel activity / detection of chemical stimulus involved in sensory perception of pain / thermoception / stereocilium bundle / channel activity / detection of mechanical stimulus involved in sensory perception of pain / calcium-release channel activity / response to pain / detection of maltose stimulus / maltose binding / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / maltose transport / maltodextrin transport / carbohydrate transmembrane transporter activity / carbohydrate transport / calcium channel activity / calcium ion transmembrane transport / transporter activity / sensory perception of pain / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / response to cold / ion transport / response to organic substance / response to organic cyclic compound / response to hydrogen peroxide / protein homotetramerization / outer membrane-bounded periplasmic space / periplasmic space / cell surface receptor signaling pathway / cellular response to DNA damage stimulus / integral component of plasma membrane / identical protein binding / plasma membrane / Transient receptor potential cation channel subfamily A member 1 / Maltose-binding periplasmic protein
Function and homology information
SourceHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 4.24 Å resolution
AuthorsPaulsen CE / Armache JP / Gao Y / Cheng Y / Julius D
CitationJournal: Nature / Year: 2015
Title: Structure of the TRPA1 ion channel suggests regulatory mechanisms.
Authors: Candice E Paulsen / Jean-Paul Armache / Yuan Gao / Yifan Cheng / David Julius
Abstract: The TRPA1 ion channel (also known as the wasabi receptor) is a detector of noxious chemical agents encountered in our environment or produced endogenously during tissue injury or drug metabolism. ...The TRPA1 ion channel (also known as the wasabi receptor) is a detector of noxious chemical agents encountered in our environment or produced endogenously during tissue injury or drug metabolism. These include a broad class of electrophiles that activate the channel through covalent protein modification. TRPA1 antagonists hold potential for treating neurogenic inflammatory conditions provoked or exacerbated by irritant exposure. Despite compelling reasons to understand TRPA1 function, structural mechanisms underlying channel regulation remain obscure. Here we use single-particle electron cryo- microscopy to determine the structure of full-length human TRPA1 to ∼4 Å resolution in the presence of pharmacophores, including a potent antagonist. Several unexpected features are revealed, including an extensive coiled-coil assembly domain stabilized by polyphosphate co-factors and a highly integrated nexus that converges on an unpredicted transient receptor potential (TRP)-like allosteric domain. These findings provide new insights into the mechanisms of TRPA1 regulation, and establish a blueprint for structure-based design of analgesic and anti-inflammatory agents.
Validation ReportPDB-ID: 3j9p

SummaryFull reportAbout validation report
DateDeposition: Feb 14, 2015 / Header (metadata) release: Apr 8, 2015 / Map release: Apr 8, 2015 / Last update: Oct 7, 2015

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 8
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-3j9p
  • Surface level: 8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_6267.map.gz (map file in CCP4 format, 105470 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
300 pix
1.22 Å/pix.
= 364.68 Å
300 pix
1.22 Å/pix.
= 364.68 Å
300 pix
1.22 Å/pix.
= 364.68 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
Contour Level:8 (by author), 8 (movie #1):
Minimum - Maximum-12.64960575 - 20.72408104
Average (Standard dev.)0E-8 (0.99999994)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions300300300
Origin-150-150-150
Limit149149149
Spacing300300300
CellA=B=C: 364.68 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21561.21561.2156
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z364.680364.680364.680
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS-150-150-150
NC/NR/NS300300300
D min/max/mean-12.65020.7240.000

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Supplemental data

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Sample components

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Entire Recombinant human TRPA1 treated with AITC

EntireName: Recombinant human TRPA1 treated with AITC / Details: The sample was monodisperse. / Number of components: 1 / Oligomeric State: tetramer
MassTheoretical: 688 kDa

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Component #1: protein, TRPA1

ProteinName: TRPA1
a.k.a: Transient receptor potential cation channel, member A1
Oligomeric Details: tetramer / Recombinant expression: Yes / Number of Copies: 4
MassTheoretical: 172 kDa / Experimental: 172 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: HEK293 GnTi-
External referencesUniProt: UniProt:O75762

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.5 mg/ml
Buffer solution: 20 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM IP6
pH: 8
Support film400 mesh holey carbon
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temperature: 120 K / Humidity: 100 % / Method: Blot for 7 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300 / Date: Jul 18, 2014
Details: Gatan K2 Summit in super-resolution counting mode. Motion correction as described in Li et al. (2013) Nature Methods.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 21 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 31000 X (nominal), 31000 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 2800 nm
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1160
Details: Every image is the average of 30 frames recorded using the K2 Summit. The final reconstruction was calculated from images motion-corrected and weighted using the method described in Scheres, 2014.
URL of raw data: http://dx.doi.org/10.6019/EMPIAR-10024
Raw dataEMPIAR-10024 (Title: Human TRPA1 ion channel with agonist AITC / Data size: 453.0 GB
Data #1: TRP ion channel dataset [picked particles - multiframe - unprocessed])

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C4 (4 fold cyclic) / Number of projections: 43585
Details: Particles were selected using an automatic selection program and manually screened. Defocus was calculated using CTFFIND3 and data were processed and refined using RELION 1.3.
3D reconstructionAlgorithm: Maximum likelihood / Software: RELION / CTF correction: Each particle / Resolution: 4.24 Å / Resolution method: FSC 0.143, gold-standard

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Atomic model buiding

Output model

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