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- PDB-3j9p: Structure of the TRPA1 ion channel determined by electron cryo-mi... -

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Entry
Database: PDB / ID: 3j9p
TitleStructure of the TRPA1 ion channel determined by electron cryo-microscopy
ComponentsMaltose-binding periplasmic protein, Transient receptor potential cation channel subfamily A member 1 chimera
KeywordsTRANSPORT PROTEIN / TRPA1 / TRP / transient / potential / receptor / ion channel / membrane protein
Function / homologyBacterial extracellular solute-binding protein / Ankyrin repeat-containing domain superfamily / Ankyrin repeat / Ion transport domain / Bacterial extracellular solute-binding protein / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Ankyrin repeat-containing domain / Transient receptor potential cation channel subfamily A member 1 / Ion transport protein ...Bacterial extracellular solute-binding protein / Ankyrin repeat-containing domain superfamily / Ankyrin repeat / Ion transport domain / Bacterial extracellular solute-binding protein / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Ankyrin repeat-containing domain / Transient receptor potential cation channel subfamily A member 1 / Ion transport protein / Ankyrin repeats (3 copies) / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding proteins, family 1 signature. / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / TRP channels / Ankyrin repeat / temperature-gated cation channel activity / thermoception / detection of chemical stimulus involved in sensory perception of pain / 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
Specimen sourceEscherichia coli (E. coli)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.24 Å resolution
AuthorsPaulsen, C.E. / Armache, J.-P. / 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 Report
SummaryFull reportAbout validation report
DateDeposition: Feb 14, 2015 / Release: Apr 8, 2015
RevisionDateData content typeGroupProviderType
1.0Apr 8, 2015Structure modelrepositoryInitial release
1.1Apr 22, 2015Structure modelDatabase references
1.2Apr 29, 2015Structure modelDatabase references

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Assembly

Deposited unit
D: Maltose-binding periplasmic protein, Transient receptor potential cation channel subfamily A member 1 chimera
A: Maltose-binding periplasmic protein, Transient receptor potential cation channel subfamily A member 1 chimera
B: Maltose-binding periplasmic protein, Transient receptor potential cation channel subfamily A member 1 chimera
C: Maltose-binding periplasmic protein, Transient receptor potential cation channel subfamily A member 1 chimera


Theoretical massNumber of molelcules
Total (without water)690,3024
Polyers690,3024
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein/peptide
Maltose-binding periplasmic protein, Transient receptor potential cation channel subfamily A member 1 chimera / Ankyrin-like with transmembrane domains protein 1 / Transformation-sensitive protein p120 / Transient Receptor Potential Ankyrin 1 ion channel


Mass: 172575.562 Da / Num. of mol.: 4 / Fragment: SEE REMARK 999 / Source: (gene. exp.) Escherichia coli, Homo sapiens / Gene: malE, ANKTM1, TRPA1 / Cell line (production host): HEK293 GnTi- / Production host: Homo sapiens (human) / References: UniProt:P0AEX9, UniProt:O75762
Sequence detailsPROTEIN IS A CHIMERA COMPRISING RESIDUES 27-392 OF UNP P0AEX9 LINKED TO RESIDUES 2-1119 OF UNP O75762.

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Recombinant human TRPA1 ion channel / Type: COMPLEX
Buffer solutionName: 20 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM IP6 / Details: 20 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM IP6 / pH: 8
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 400 mesh holey carbon
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temp: 120 K / Humidity: 100 %
Details: Blot for 7 seconds before plunging into liquid ethane (FEI VITROBOT MARK I).
Method: Blot for 7 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyMicroscope model: 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 / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 31000 / Calibrated magnification: 31000 / Nominal defocus max: 2800 nm / Nominal defocus min: 1500 nm / Cs: 2 mm
Specimen holderTemperature: 120 kelvins
Image recordingElectron dose: 21 e/Å2
Details: Gatan K2 Summit in super-resolution counting mode. Motion correction as described in Li et al. (2013) Nature Methods.
Film or detector model: GATAN K2 (4k x 4k)
Image scansNumber digital images: 1160
RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m l: M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM softwareName: Relion / Category: 3D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: C4
3D reconstructionMethod: Maximum likelihood / Resolution: 4.24 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 43585 / Nominal pixel size: 1.2156 / Actual pixel size: 1.2156 / Symmetry type: POINT
Number of atoms included #LASTProtein: 16952 / Nucleic acid: 0 / Ligand: 0 / Solvent: 0 / Total: 16952

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