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- PDB-6nr2: Cryo-EM structure of the TRPM8 ion channel in complex with the me... -

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Basic information

Entry
Database: PDB / ID: 6nr2
TitleCryo-EM structure of the TRPM8 ion channel in complex with the menthol analog WS-12 and PI(4,5)P2
ComponentsTransient receptor potential cation channel subfamily M member 8
KeywordsTRANSPORT PROTEIN / ion 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
Function / homologyChem-KXP / Chem-KXS
Function and homology information
Biological speciesFicedula albicollis (Collared flycatcher)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsYin, Y. / Le, S.C. / Hsu, A.L. / Borgnia, M.J. / Yang, H. / Lee, S.-Y.
Funding support United States, 1items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 20, 2019Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2019Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.journal_volume / _citation_author.identifier_ORCID ..._citation.journal_volume / _citation_author.identifier_ORCID / _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.country / _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: Transient receptor potential cation channel subfamily M member 8
B: Transient receptor potential cation channel subfamily M member 8
C: Transient receptor potential cation channel subfamily M member 8
D: Transient receptor potential cation channel subfamily M member 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)525,66012
Polymers520,3144
Non-polymers5,3468
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 130078.453 Da / Num. of mol.: 4 / Mutation: F535A,Y538D,Y539D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ficedula albicollis (Collared flycatcher)
Gene: TRPM8 / Production host: Homo sapiens (human)
#2: Chemical
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


Mass: 1047.088 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C47H85O19P3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-KXS / (1R,2S,5R)-N-(4-methoxyphenyl)-5-methyl-2-(propan-2-yl)cyclohexane-1-carboxamide


Mass: 289.412 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C18H27NO2 / Feature type: SUBJECT OF INVESTIGATION
Compound details1
Sequence detailsThe complete sample sequence including tags and engineered mutations is the following: ...The complete sample sequence including tags and engineered mutations is the following:MATLFQVSMGSMRHRRNGNFESSRLLYSSMSRSIDVACSDADLANFIQENFKKRECVFFTKD TKSMGNLCKCGYPENQHIEGTQVNTTEKWNYKKHTKELPTDAFGDIQFENLGKRGKYIRL SCDTDSETLYDLMTQHWHLKTPNLVISVTGGAKNFALKPRMRKIFSRLIYIAQSKGAWIF TGGTHYGLMKYIGEVVRDNTISRSSEENVVAIGIAAWGMISNRETLIRTADSDGSYLAHY IMDDLKRDPLYCLDNNHTHLLLVDNGTHGHPTIEAKVRTQLEKYISERVIPESNYGGKIP IVCFAQGGGKETLKSINVAIKSKIPCVVVEGSGRIADVIASLVEAEGTLASSCVKESLLR FLPRTISRLSEEETESWIKWIKEVLESPHLLTVIKIEEAGDEIVSNAISFALYKAFSTNE HDRDNWNGQLKLLLEWNQLDLASDEIFTNDRNWESADLQDVMFTALVKDRPKFVRLFLEN GLNLRKFLTTEVLRELYTNNFSSLVFKNLQIAKNSYNDALLTFVWKMVEDFRRGAKRDDK NSKDEMEIELSEECPITRHPLQALFIWSVLQNKKELSKVIWEQTRGCTLAALGASKLLKS MAKVKNDINAAGESEELANEYETRAVELFTECYSNDEDLAEQLLTYSCEAWGGSNCLELA VEARDQQFIAQPGVQNFLSKQWYGEISRDTKNWKIILCLFFFPLIGCGFISFRKKPVEKT KKLFLYYVSFFTSPFVVFSWNVIFYIAFLLLFAYVLLMDFQKEPTALEIILYVLVFILLC DEVRQWYMNGSKYFSDLWNVMDTLAIFYFIAGIVFRLHSDESSWYSGRVIFCLDYIVFTL RLIHIFTVSRNLGPKIIMLQRMMIDVFFFLFLFAVWMVAFGVARQGILRKNEHRWEWIFR SVIYEPYLAMFGQYPDDIDGTTYNFDHCTFSGNESKPLCVELDANNQPRFPEWITIPLVC IYMLSTNILLVNLLVAMFGYTVGSVQENNDQVWKFQRFFLVQEYCSRLTIPFPFVIFAYI FMVMRKCFKCCCKKESKEPSVCCSRNEDNEILAWEAVMKENYLVKINTKASDSSEEMVHR FRQLDAKLSDLKGLLKEISSKIKSNSLEVLFQGPDYKDDDDKAHHHHHHHHHH

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Transient receptor potential melastatin member 8 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Ficedula albicollis (Collared flycatcher)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1250 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 42 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4006

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3particle selection
2EPUimage acquisitionAutomated data acquisition
4RELION3CTF correction
5Gctf1.18CTF correction
8Coot0.8.8model fitting
10RELION3initial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
20PHENIX1.12-2829model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1622434
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 46940 / Symmetry type: POINT
Atomic model buildingB value: 100 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6BPQ

6bpq

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