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- EMDB-21538: Structure of TRPA1 (ligand-free and calcium-free), PMAL-C8, class 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-21538
TitleStructure of TRPA1 (ligand-free and calcium-free), PMAL-C8, class 2
Map data
SampleTRPA1 (ligand-free and calcium free), solubilized in PMAL-C8, class 2:
Transient receptor potential A1 (TRPA1)
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsZhao J / Lin King JV / Paulsen CE / Cheng Y / Julius D
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R35 NS105038 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM098672 United States
CitationJournal: Nature / Year: 2020
Title: Irritant-evoked activation and calcium modulation of the TRPA1 receptor.
Authors: Jianhua Zhao / John V Lin King / Candice E Paulsen / Yifan Cheng / David Julius /
Abstract: The transient receptor potential ion channel TRPA1 is expressed by primary afferent nerve fibres, in which it functions as a low-threshold sensor for structurally diverse electrophilic irritants, ...The transient receptor potential ion channel TRPA1 is expressed by primary afferent nerve fibres, in which it functions as a low-threshold sensor for structurally diverse electrophilic irritants, including small volatile environmental toxicants and endogenous algogenic lipids. TRPA1 is also a 'receptor-operated' channel whose activation downstream of metabotropic receptors elicits inflammatory pain or itch, making it an attractive target for novel analgesic therapies. However, the mechanisms by which TRPA1 recognizes and responds to electrophiles or cytoplasmic second messengers remain unknown. Here we use strutural studies and electrophysiology to show that electrophiles act through a two-step process in which modification of a highly reactive cysteine residue (C621) promotes reorientation of a cytoplasmic loop to enhance nucleophilicity and modification of a nearby cysteine (C665), thereby stabilizing the loop in an activating configuration. These actions modulate two restrictions controlling ion permeation, including widening of the selectivity filter to enhance calcium permeability and opening of a canonical gate at the cytoplasmic end of the pore. We propose a model to explain functional coupling between electrophile action and these control points. We also characterize a calcium-binding pocket that is highly conserved across TRP channel subtypes and accounts for all aspects of calcium-dependent TRPA1 regulation, including potentiation, desensitization and activation by metabotropic receptors. These findings provide a structural framework for understanding how a broad-spectrum irritant receptor is controlled by endogenous and exogenous agents that elicit or exacerbate pain and itch.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionMar 11, 2020-
Header (metadata) releaseApr 8, 2020-
Map releaseMay 6, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21538.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.22 Å/pix.
x 256 pix.
= 311.194 Å
1.22 Å/pix.
x 256 pix.
= 311.194 Å
1.22 Å/pix.
x 256 pix.
= 311.194 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 LevelBy AUTHOR: 4 / Movie #1: 4
Minimum - Maximum-14.989781 - 22.766779
Average (Standard dev.)-0.00000000277 (±1)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 311.1936 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21560156251.21560156251.2156015625
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z311.194311.194311.194
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-14.99022.767-0.000

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Supplemental data

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Segmentation: #1

Fileemd_21538_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_21538_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_21538_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire TRPA1 (ligand-free and calcium free), solubilized in PMAL-C8, class 2

EntireName: TRPA1 (ligand-free and calcium free), solubilized in PMAL-C8, class 2
Number of components: 2

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Component #1: protein, TRPA1 (ligand-free and calcium free), solubilized in PMA...

ProteinName: TRPA1 (ligand-free and calcium free), solubilized in PMAL-C8, class 2
Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: HEK293F

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Component #2: protein, Transient receptor potential A1 (TRPA1)

ProteinName: Transient receptor potential A1 (TRPA1) / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 8
Support filmunspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C4 (4 fold cyclic) / Number of projections: 28558
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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