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- EMDB-15982: Human apo TRPM8 in a closed state (consensus map) -

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Basic information

Entry
Database: EMDB / ID: EMD-15982
TitleHuman apo TRPM8 in a closed state (consensus map)
Map datamap sharpened locally with Local Filtering tool in cryoSPARC
Sample
  • Complex: Human TRPM8 in apo form
    • Protein or peptide: TRPM8
Keywordscalcium ion channel / cold temperature sensor / MEMBRANE PROTEIN
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
Transient receptor potential cation channel subfamily M member 8
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.65 Å
AuthorsPalchevskyi S / Czarnocki-Cieciura M / Vistoli G / Gervasoni S / Nowak E / Beccari AR / Nowotny M / Talarico C
Funding support Poland, 1 items
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, 2022-
Header (metadata) releaseNov 1, 2023-
Map releaseNov 1, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15982.png

Downloads & links

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Map

FileDownload / File: emd_15982.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap sharpened locally with Local Filtering tool in cryoSPARC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 420 pix.
= 344.4 Å		0.82 Å/pix.
x 420 pix.
= 344.4 Å		0.82 Å/pix.
x 420 pix.
= 344.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-3.1724246 - 5.208554
Average (Standard dev.)0.0041521587 (±0.08090575)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 344.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15982_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: raw map

Fileemd_15982_additional_1.map
Annotationraw map
Projections & Slices
AxesZYX

Projections

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Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_15982_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_15982_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human TRPM8 in apo form

EntireName: Human TRPM8 in apo form
Components
  • Complex: Human TRPM8 in apo form
    • Protein or peptide: TRPM8

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Supramolecule #1: Human TRPM8 in apo form

SupramoleculeName: Human TRPM8 in apo form / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 510 KDa

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Macromolecule #1: TRPM8

MacromoleculeName: TRPM8 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: SNSFRAARLS MRNRRNDTLD STRTLYSSAS RSTDLSYSES DLVNFIQANF KKRECVFFTK DSKATENVCK CGYAQSQHME GTQINQSEKW NYKKHTKEFP TDAFGDIQFE TLGKKGKYIR LSCDTDAEIL YELLTQHWHL KTPNLVISVT GGAKNFALKP RMRKIFSRLI ...String:
SNSFRAARLS MRNRRNDTLD STRTLYSSAS RSTDLSYSES DLVNFIQANF KKRECVFFTK DSKATENVCK CGYAQSQHME GTQINQSEKW NYKKHTKEFP TDAFGDIQFE TLGKKGKYIR LSCDTDAEIL YELLTQHWHL KTPNLVISVT GGAKNFALKP RMRKIFSRLI YIAQSKGAWI LTGGTHYGLM KYIGEVVRDN TISRSSEENI VAIGIAAWGM VSNRDTLIRN CDAEGYFLAQ YLMDDFTRDP LYILDNNHTH LLLVDNGCHG HPTVEAKLRN QLEKYISERT IQDSNYGGKI PIVCFAQGGG KETLKAINTS IKNKIPCVVV EGSGQIADVI ASLVEVEDAL TSSAVKEKLV RFLPRTVSRL PEEETESWIK WLKEILECSH LLTVIKMEEA GDEIVSNAIS YALYKAFSTS EQDKDNWNGQ LKLLLEWNQL DLANDEIFTN DRRWESADLQ EVMFTALIKD RPKFVRLFLE NGLNLRKFLT HDVLTELFSN HFSTLVYRNL QIAKNSYNDA LLTFVWKLVA NFRRGFRKED RNGRDEMDIE LHDVSPITRH PLQALFIWAI LQNKKELSKV IWEQTRGCTL AALGASKLLK TLAKVKNDIN AAGESEELAN EYETRAVELF TECYSSDEDL AEQLLVYSCE AWGGSNCLEL AVEATDQHFI AQPGVQNFLS KQWYGEISRD TKNWKIILCL FIIPLVGCGF VSFRKKPVDK HKKLLWYYVA FFTSPFVVFS WNVVFYIAFL LLFAYVLLMD FHSVPHPPEL VLYSLVFVLF CDEVRQWYVN GVNYFTDLWN VMDTLGLFYF IAGIVFRLHS SNKSSLYSGR VIFCLDYIIF TLRLIHIFTV SRNLGPKIIM LQRMLIDVFF FLFLFAVWMV AFGVARQGIL RQNEQRWRWI FRSVIYEPYL AMFGQVPSDV DGTTYDFAHC TFTGNESKPL CVELDEHNLP RFPEWITIPL VCIYMLSTNI LLVNLLVAMF GYTVGTVQEN NDQVWKFQRY FLVQEYCSRL NIPFPFIVFA YFYMVVKKCF KCCCKEKNME SSVCCFKNED NETLAWEGVM KENYLVKINT KANDTSEEMR HRFRQLDTKL NDLKGLLKEI ANKIK

UniProtKB: Transient receptor potential cation channel subfamily M member 8

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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: 7.4
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
Details: The grid was glow-discharged from both sides prior to vitrification
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Detailsprotein solubilized by LMNG

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 7918 / Average electron dose: 41.42 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 105000
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: 1239855
Startup modelType of model: OTHER / Details: cryoSPARC Ab-Initio reconstruction
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.65 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 110176
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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