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- EMDB-5334: TRPA1 channel structure at 16A resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-5334
TitleTRPA1 channel structure at 16A resolution
Map dataThis is a TRPA1 channel map at 16A.
Sample
  • Sample: TRPA1 channel
  • Protein or peptide: transient receptor potential cation channel subfamily A member 1
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 16.0 Å
AuthorsCvetkov TL / Huynh KW / Cohen MR / Moiseenkova-Bell VY
CitationJournal: J Biol Chem / Year: 2011
Title: Molecular architecture and subunit organization of TRPA1 ion channel revealed by electron microscopy.
Authors: Teresa L Cvetkov / Kevin W Huynh / Matthew R Cohen / Vera Y Moiseenkova-Bell /
Abstract: Transient receptor potential ankyrin 1 (TRPA1) is a non-selective ion channel, which is expressed in nociceptor sensory neurons and transduces chemical, inflammatory, and neuropathic pain signals. ...Transient receptor potential ankyrin 1 (TRPA1) is a non-selective ion channel, which is expressed in nociceptor sensory neurons and transduces chemical, inflammatory, and neuropathic pain signals. Numerous non-reactive compounds and electrophilic compounds, such as endogenous inflammatory mediators and exogenous pungent chemicals, can activate TRPA1. Here we report a 16-Å resolution structure of purified, functional, amphipol-stabilized TRPA1 analyzed by single-particle EM. Molecular models of the N and C termini of the channel were generated using the I-TASSER protein structure prediction server and docked into the EM density to provide insight into the TRPA1 subunit organization. This structural analysis suggests a location for critical N-terminal cysteine residues involved in electrophilic activation at the interface between neighboring subunits. Our results indicate that covalent modifications within this pocket may alter interactions between subunits and promote conformational changes that lead to channel activation.
History
DepositionAug 29, 2011-
Header (metadata) releaseSep 6, 2011-
Map releaseSep 13, 2011-
UpdateSep 13, 2011-
Current statusSep 13, 2011Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 9.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 9.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5334_1.png

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Map

FileDownload / File: emd_5334.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a TRPA1 channel map at 16A.
Voxel sizeX=Y=Z: 3.4 Å
Density
Contour LevelBy AUTHOR: 9.199999999999999 / Movie #1: 9.2
Minimum - Maximum-11.9779 - 22.479700000000001
Average (Standard dev.)0.055064 (±1.58362)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-80-80
Dimensions160160160
Spacing160160160
CellA=B=C: 544 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.43.43.4
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z544.000544.000544.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-80-80-80
NC/NR/NS160160160
D min/max/mean-11.97822.4800.055

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Supplemental data

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Sample components

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Entire : TRPA1 channel

EntireName: TRPA1 channel
Components
  • Sample: TRPA1 channel
  • Protein or peptide: transient receptor potential cation channel subfamily A member 1

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Supramolecule #1000: TRPA1 channel

SupramoleculeName: TRPA1 channel / type: sample / ID: 1000 / Details: The sample was monodisperse. / Oligomeric state: Tetramer / Number unique components: 1
Molecular weightExperimental: 536 KDa / Theoretical: 525 KDa / Method: Gel filtration

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Macromolecule #1: transient receptor potential cation channel subfamily A member 1

MacromoleculeName: transient receptor potential cation channel subfamily A member 1
type: protein_or_peptide / ID: 1 / Name.synonym: TRPA1 / Number of copies: 1 / Oligomeric state: Tetramer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: house mouse / Location in cell: Plasma Membrane
Molecular weightExperimental: 536 KDa / Theoretical: 525 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: YeP

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 8
Details: 20 mM HEPES, pH 8.0, 150 mM NaCl, 10% glycerol, 1.0 mM DTT
StainingType: NEGATIVE
Details: TRPA1 was adsorbed on carbon-film coated copper grids, washed with 3 droplets of pure water and subsequently stained with 1% uranyl acetate.
GridDetails: 400 mesh copper grids with carbon
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 55000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 200,000 times magnification
DateMay 12, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 20 e/Å2
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each image
Final two d classificationNumber classes: 218
Final reconstructionResolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 / Number images used: 8505

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