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- PDB-8bdc: Human apo TRPM8 in a closed state (composite map) -

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Basic information

Entry
Database: PDB / ID: 8bdc
TitleHuman apo TRPM8 in a closed state (composite map)
ComponentsTransient receptor potential cation channel subfamily M member 8
KeywordsMEMBRANE PROTEIN / calcium ion channel / cold temperature sensor
Function / homology
Function and homology information


ligand-gated calcium channel activity / thermoception / TRP channels / response to cold / calcium ion transmembrane transport / calcium channel activity / intracellular calcium ion homeostasis / positive regulation of cold-induced thermogenesis / membrane raft / external side of plasma membrane ...ligand-gated calcium channel activity / thermoception / TRP channels / response to cold / calcium ion transmembrane transport / calcium channel activity / intracellular calcium ion homeostasis / positive regulation of cold-induced thermogenesis / membrane raft / external side of plasma membrane / endoplasmic reticulum membrane / identical protein binding / plasma membrane
Similarity search - Function
TRPM, SLOG domain / SLOG in TRPM / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Chem-POV / UNDECANE / CHOLESTEROL HEMISUCCINATE / Transient receptor potential cation channel subfamily M member 8
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.65 Å
AuthorsPalchevskyi, S. / Czarnocki-Cieciura, M. / Vistoli, G. / Gervasoni, S. / Nowak, E. / Beccari, A.R. / Nowotny, M. / Talarico, C.
Funding support Poland, 1items
OrganizationGrant numberCountry
Other government Poland
CitationJournal: Commun Biol / Year: 2023
Title: Structure of human TRPM8 channel.
Authors: Sergii Palchevskyi / Mariusz Czarnocki-Cieciura / Giulio Vistoli / Silvia Gervasoni / Elżbieta Nowak / Andrea R Beccari / Marcin Nowotny / Carmine Talarico /
Abstract: TRPM8 is a non-selective cation channel permeable to both monovalent and divalent cations that is activated by multiple factors, such as temperature, voltage, pressure, and changes in osmolality. It ...TRPM8 is a non-selective cation channel permeable to both monovalent and divalent cations that is activated by multiple factors, such as temperature, voltage, pressure, and changes in osmolality. It is a therapeutic target for anticancer drug development, and its modulators can be utilized for several pathological conditions. Here, we present a cryo-electron microscopy structure of a human TRPM8 channel in the closed state that was solved at 2.7 Å resolution. Our structure comprises the most complete model of the N-terminal pre-melastatin homology region. We also visualized several lipids that are bound by the protein and modeled how the human channel interacts with icilin. Analyses of pore helices in available TRPM structures showed that all these structures can be grouped into different closed, desensitized and open state conformations based on the register of the pore helix S6 which positions particular amino acid residues at the channel constriction.
History
DepositionOct 19, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 1, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)519,36228
Polymers511,6194
Non-polymers7,74324
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "B"
d_2ens_1chain "A"
d_3ens_1chain "C"
d_4ens_1chain "D"

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11LEULEULYSLYSBB41 - 110442 - 1105
d_12Y01Y01Y01Y01BK1201
d_13POVPOVPOVPOVBL1202
d_14Y01Y01Y01Y01BM1203
d_15UNDUNDUNDUNDBN1204
d_16NANANANABO - P1205 - 1206
d_21LEULEULYSLYSAA41 - 110442 - 1105
d_22Y01Y01Y01Y01AE1201
d_23POVPOVPOVPOVAF1202
d_24Y01Y01Y01Y01AG1203
d_25UNDUNDUNDUNDAH1204
d_26NANANANAAI - J1205 - 1206
d_31LEULEULYSLYSCC41 - 110442 - 1105
d_32Y01Y01Y01Y01CQ1201
d_33POVPOVPOVPOVCR1202
d_34Y01Y01Y01Y01CS1203
d_35UNDUNDUNDUNDCT1204
d_36NANANANACU - V1205 - 1206
d_41LEULEULYSLYSDD41 - 110442 - 1105
d_42Y01Y01Y01Y01DW1201
d_43POVPOVPOVPOVDX1202
d_44Y01Y01Y01Y01DY1203
d_45UNDUNDUNDUNDDZ1204
d_46NANANANADAA - BA1205 - 1206

NCS oper:
IDCodeMatrixVector
1given(-3.14610062847E-5, -0.99999999321, -0.00011220549263), (0.999999903076, -3.15102787651E-5, 0.000439152439641), (-0.000439155972286, -0.000112191665577, 0.999999897278)344.427818719, -0.0981232820821, 0.0904883551178
2given(-0.99999969958, 1.68812178946E-5, -0.000774955053208), (-1.72014753259E-5, -0.999999914462, 0.00041325455754), (-0.00077494801068, 0.00041326776376, 0.999999614333)344.556012019, 344.305239442, 0.0601380837307
3given(2.28190235192E-5, 0.999999875498, -0.000498481048793), (-0.999999990507, 2.27512831081E-5, -0.000135898901949), (-0.000135887543946, 0.000498484145141, 0.999999866524)0.109098468249, 344.414758605, -0.0764299010179

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Components

#1: Protein
Transient receptor potential cation channel subfamily M member 8 / Long transient receptor potential channel 6 / LTrpC-6 / LTrpC6 / Transient receptor potential p8 / Trp-p8


Mass: 127904.719 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TRPM8, LTRPC6, TRPP8 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q7Z2W7
#2: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C31H50O4
#3: Chemical
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC / POPC


Mass: 760.076 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
#4: Chemical
ChemComp-UND / UNDECANE / LIPID FRAGMENT / Undecane


Mass: 156.308 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C11H24
#5: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Na
Has ligand of interestN

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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: Human TRPM8 in apo form / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.51 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf9
Buffer solutionpH: 7.4
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: protein solubilized by LMNG
Specimen supportDetails: The grid was glow-discharged from both sides prior to vitrification
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 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: 105000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 41.42 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 7918
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refinedev_4694refinement
PHENIXdev_4694refinement
EM software
IDNameVersionCategoryDetails
1crYOLO1.7.6particle selection
2EPU2.10.0.5RELimage acquisition
4CTFFIND4.1CTF correction
7Coot0.9.8.1model fitting
9PHENIX1.2model refinementdev-4694
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARCfinal Euler assignment
12RELION3.1classification
13cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1239855
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110176
Details: composite map created by combining consensus map with four copies of focused pre-MHR + MHR1/2 map
Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 54.85 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002430856
ELECTRON MICROSCOPYf_angle_d0.386642016
ELECTRON MICROSCOPYf_chiral_restr0.03594856
ELECTRON MICROSCOPYf_plane_restr0.00285180
ELECTRON MICROSCOPYf_dihedral_angle_d5.60784476
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints1.66441766482E-12
ens_1d_3CELECTRON MICROSCOPYNCS constraints4.95718522276E-10
ens_1d_4DELECTRON MICROSCOPYNCS constraints3.57748926671E-11

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