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- EMDB-0488: Cryo-EM structure of the TRPM8 ion channel in complex with high o... -

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Basic information

Entry
Database: EMDB / ID: EMD-0488
TitleCryo-EM structure of the TRPM8 ion channel in complex with high occupancy icilin, PI(4,5)P2, and calcium
Map dataem-volume_P1
Sample
  • Complex: Transient receptor potential melastatin member 8
    • Protein or peptide: Transient receptor potential cation channel subfamily M member 8
  • Ligand: Icilin
  • Ligand: (2S)-1-{[(R)-hydroxy{[(1R,2R,3S,4R,5R,6S)-2,3,6-trihydroxy-4,5-bis(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}-3-(octadecanoyloxy)propan-2-yl icosa-5,8,11,14-tetraenoate
  • Ligand: CALCIUM ION
Keywordsion channel / TRP channel / TRPM channel / TRPM8 channel / cold sensing / lipid sensing / menthol / icilin / WS-12 / PI(4 / 5)P2 / cooling agent / MEMBRANE PROTEIN / calcium-permeable ion channel / TRANSPORT PROTEIN
Biological speciesFicedula albicollis (Collared flycatcher)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsYin Y / Le SC
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) United States
CitationJournal: Science / Year: 2019
Title: Structural basis of cooling agent and lipid sensing by the cold-activated TRPM8 channel.
Authors: Ying Yin / Son C Le / Allen L Hsu / Mario J Borgnia / Huanghe Yang / Seok-Yong Lee /
Abstract: Transient receptor potential melastatin member 8 (TRPM8) is a calcium ion (Ca)-permeable cation channel that serves as the primary cold and menthol sensor in humans. Activation of TRPM8 by cooling ...Transient receptor potential melastatin member 8 (TRPM8) is a calcium ion (Ca)-permeable cation channel that serves as the primary cold and menthol sensor in humans. Activation of TRPM8 by cooling compounds relies on allosteric actions of agonist and membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP), but lack of structural information has thus far precluded a mechanistic understanding of ligand and lipid sensing by TRPM8. Using cryo-electron microscopy, we determined the structures of TRPM8 in complex with the synthetic cooling compound icilin, PIP, and Ca, as well as in complex with the menthol analog WS-12 and PIP Our structures reveal the binding sites for cooling agonists and PIP in TRPM8. Notably, PIP binds to TRPM8 in two different modes, which illustrate the mechanism of allosteric coupling between PIP and agonists. This study provides a platform for understanding the molecular mechanism of TRPM8 activation by cooling agents.
History
DepositionJan 22, 2019-
Header (metadata) releaseFeb 6, 2019-
Map releaseFeb 20, 2019-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nr3
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0488.png

Downloads & links

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Map

FileDownload / File: emd_0488.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationem-volume_P1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.109554574 - 0.22814246
Average (Standard dev.)0.000039037095 (±0.0069128843)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z276.480276.480276.480
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1100.2280.000

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Supplemental data

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Half map: em-half-volume P1

Fileemd_0488_half_map_1.map
Annotationem-half-volume_P1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: em-half-volume P2

Fileemd_0488_half_map_2.map
Annotationem-half-volume_P2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Transient receptor potential melastatin member 8

EntireName: Transient receptor potential melastatin member 8
Components
  • Complex: Transient receptor potential melastatin member 8
    • Protein or peptide: Transient receptor potential cation channel subfamily M member 8
  • Ligand: Icilin
  • Ligand: (2S)-1-{[(R)-hydroxy{[(1R,2R,3S,4R,5R,6S)-2,3,6-trihydroxy-4,5-bis(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}-3-(octadecanoyloxy)propan-2-yl icosa-5,8,11,14-tetraenoate
  • Ligand: CALCIUM ION

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Supramolecule #1: Transient receptor potential melastatin member 8

SupramoleculeName: Transient receptor potential melastatin member 8 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Ficedula albicollis (Collared flycatcher)

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Macromolecule #1: Transient receptor potential cation channel subfamily M member 8

MacromoleculeName: Transient receptor potential cation channel subfamily M member 8
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Ficedula albicollis (Collared flycatcher)
Molecular weightTheoretical: 130.011391 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MATLFQVSMG SMRHRRNGNF ESSRLLYSSM SRSIDVACSD ADLANFIQEN FKKRECVFFT KDTKSMGNLC KCGYPENQHI EGTQVNTTE KWNYKK(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) ...String:
MATLFQVSMG SMRHRRNGNF ESSRLLYSSM SRSIDVACSD ADLANFIQEN FKKRECVFFT KDTKSMGNLC KCGYPENQHI EGTQVNTTE KWNYKK(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)K YIRLSCDTDS ETLYDLMTQH WHLKTPNLVI SVTG GAKNF ALKPRMRKIF SRLIYIAQSK GAWIFTGGTH YGLMKYIGEV VRDNTISRSS EENVVAIGIA AWGMISNRET LIRTA DSDG SYLAHYIMDD LKRDPLYCLD NNHTHLLLVD NGTHGHPTIE AKVRTQLEKY ISERVIPESN YGGKIPIVCF AQGGGK ETL KSINVAIKSK IPCVVVEGSG RIADVIASLV EAEGTLASSC VKESLLRFLP RTISRLSEEE TESWIKWIKE VLESPHL LT VIKIEEAGDE IVSNAISFAL YKAFSTNEHD RDNWNGQLKL LLEWNQLDLA SDEIFTNDRN WESADLQDVM FTALVKDR P KFVRLFLENG LNLRKFLTTE VLRELYTNNF SSLVFKNLQI AKNSYNDALL TFVWKMVEDF RRGAKRDDKN SKDEMEIEL SEECPITRHP LQALFIWSVL QNKKELSKVI WEQTRGCTLA ALGASKLLKS MAKVKNDINA AGESEELANE YETRAVELFT ECYSNDEDL AEQLLTYSCE AWGGSNCLEL AVEARDQQFI AQPGVQNFLS KQWYGEISRD TKNWKIILCL FFFPLIGCGF I SFRKKPVE KTKKLFLYYV SFFTSPFVVF SWNVIFYIAF LLLFAYVLLM DFQKEPTALE IILYVLVFIL LCDEVRQWYM NG SKYFSDL WNVMDTLGIF YFIAGIVFRL HSDESSWYSG RVIFCLDYIV FTLRLIHIFT VSRNLGPKII MLQRMMIDVF FFL FLFAVW MVAFGVARQG ILRKNEHRWE WIFRSVIYEP YLAMFGQYPD DIDGTTYNFD HCTFSGNESK PLCVELDANN QPRF PEWIT IPLVCIYMLS TNILLVNLLV AMFGYTVGSV QENNDQVWKF QRFFLVQEYC SRLTIPFPFV IFAYIFMVMR KCFKC CCKK ESKEPSVCCS RNEDNEILAW EAVMKENYLV KINTKASDSS EE(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)KI KSNSLEVLFQ GPDYKDDDDK AHHHHHHHHH H

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Macromolecule #2: Icilin

MacromoleculeName: Icilin / type: ligand / ID: 2 / Number of copies: 4 / Formula: KX7
Molecular weightTheoretical: 311.292 Da
Chemical component information

ChemComp-KX7:
Icilin / agonist*YM

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Macromolecule #3: (2S)-1-{[(R)-hydroxy{[(1R,2R,3S,4R,5R,6S)-2,3,6-trihydroxy-4,5-bi...

MacromoleculeName: (2S)-1-{[(R)-hydroxy{[(1R,2R,3S,4R,5R,6S)-2,3,6-trihydroxy-4,5-bis(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}-3-(octadecanoyloxy)propan-2-yl icosa-5,8,11,14-tetraenoate
type: ligand / ID: 3 / Number of copies: 4 / Formula: KXP
Molecular weightTheoretical: 1.047088 KDa
Chemical component information

ChemComp-KXP:
(2S)-1-{[(R)-hydroxy{[(1R,2R,3S,4R,5R,6S)-2,3,6-trihydroxy-4,5-bis(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}-3-(octadecanoyloxy)propan-2-yl icosa-5,8,11,14-tetraenoate

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

Concentration0.5 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 26 sec. / Details: 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3705 / Average exposure time: 60.0 sec. / Average electron dose: 42.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.25 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1115545
Startup modelType of model: EMDB MAP
EMDB ID:

7127


Details: The EM map for the previously reported apo TRPM8 structure (EMD-7127) was low-pass filtered to 30-Angstrom and used as an initial model without a reference mask.
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 101192
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.0)

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Atomic model buiding 1

Initial modelPDB ID:

6bpq


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 100
Output model

PDB-6nr3:
Cryo-EM structure of the TRPM8 ion channel in complex with high occupancy icilin, PI(4,5)P2, and calcium

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