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- EMDB-21131: Structure of TRPA1 treated with A-967079 (class 2), PMAL-C8 -

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Basic information

Entry
Database: EMDB / ID: EMD-21131
TitleStructure of TRPA1 treated with A-967079 (class 2), PMAL-C8
Map dataFull map
Sample
  • Complex: TRPA1 treated with A-967079 (but ligand not bound), solubilized in PMAL-C8
    • Protein or peptide: TRPA1
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 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.
History
DepositionDec 16, 2019-
Header (metadata) releaseJan 15, 2020-
Map releaseMay 6, 2020-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

emd_21131.png

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Map

FileDownload / File: emd_21131.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map
Voxel sizeX=Y=Z: 1.2156 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.3
Minimum - Maximum-0.5473419 - 1.2317989
Average (Standard dev.)0.0052205874 (±0.051233448)
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/NZ172172172
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.5471.2320.005

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Supplemental data

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Mask #1

Fileemd_21131_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 1

Fileemd_21131_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_21131_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

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Sample components

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Entire : TRPA1 treated with A-967079 (but ligand not bound), solubilized i...

EntireName: TRPA1 treated with A-967079 (but ligand not bound), solubilized in PMAL-C8
Components
  • Complex: TRPA1 treated with A-967079 (but ligand not bound), solubilized in PMAL-C8
    • Protein or peptide: TRPA1

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Supramolecule #1: TRPA1 treated with A-967079 (but ligand not bound), solubilized i...

SupramoleculeName: TRPA1 treated with A-967079 (but ligand not bound), solubilized in PMAL-C8
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293F

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Macromolecule #1: TRPA1

MacromoleculeName: TRPA1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKRSLRKMWR PGEKKEPQGV VYEDVPDDTE DFKESLKVVF EGSAYGLQNF NKQKKLKRCD DMDTFFLHYA AAEGQIELME KITRDSSLEV LHEMDDYGNT PLHCAVEKNQ IESVKFLLSR GANPNLRNFN MMAPLHIAVQ GMNNEVMKVL LEHRTIDVNL EGENGNTAVI ...String:
MKRSLRKMWR PGEKKEPQGV VYEDVPDDTE DFKESLKVVF EGSAYGLQNF NKQKKLKRCD DMDTFFLHYA AAEGQIELME KITRDSSLEV LHEMDDYGNT PLHCAVEKNQ IESVKFLLSR GANPNLRNFN MMAPLHIAVQ GMNNEVMKVL LEHRTIDVNL EGENGNTAVI IACTTNNSEA LQILLKKGAK PCKSNKWGCF PIHQAAFSGS KECMEIILRF GEEHGYSRQL HINFMNNGKA TPLHLAVQNG DLEMIKMCLD NGAQIDPVEK GRCTAIHFAA TQGATEIVKL MISSYSGSVD IVNTTDGCHE TMLHRASLFD HHELADYLIS VGADINKIDS EGRSPLILAT ASASWNIVNL LLSKGAQVDI KDNFGRNFLH LTVQQPYGLK NLRPEFMQMQ QIKELVMDED NDGCTPLHYA CRQGGPGSVN NLLGFNVSIH SKSKDKKSPL HFAASYGRIN TCQRLLQDIS DTRLLNEGDL HGMTPLHLAA KNGHDKVVQL LLKKGALFLS DHNGWTALHH ASMGGYTQTM KVILDTNLKC TDRLDEDGNT ALHFAAREGH AKAVALLLSH NADIVLNKQQ ASFLHLALHN KRKEVVLTII RSKRWDECLK IFSHNSPGNK CPITEMIEYL PECMKVLLDF CMLHSTEDKS CRDYYIEYNF KYLQCPLEFT KKTPTQDVIY EPLTALNAMV QNNRIELLNH PVCKEYLLMK WLAYGFRAHM MNLGSYCLGL IPMTILVVNI KPGMAFNSTG IINETSDHSE ILDTTNSYLI KTCMILVFLS SIFGYCKEAG QIFQQKRNYF MDISNVLEWI IYTTGIIFVL PLFVEIPAHL QWQCGAIAVY FYWMNFLLYL QRFENCGIFI VMLEVILKTL LRSTVVFIFL LLAFGLSFYI LLNLQDPFSS PLLSIIQTFS MMLGDINYRE SFLEPYLRNE LAHPVLSFAQ LVSFTIFVPI VLMNLLIGLA VGDIADVQKH ASLKRIAMQV ELHTSLEKKL PLWFLRKVDQ KSTIVYPNKP RSGGMLFHIF CFLFCTGEIR QEIPNADKSL EMEILKQKYR LKDLTFLLEK QHELIKLIIQ KMEIISETED DDSHCSFQDR FKKEQMEQRN SRWNTVLRAV KAKTHHLEP

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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

BufferpH: 8
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 78959
FSC plot (resolution estimation)

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