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- PDB-6d73: Cryo-EM structure of the zebrafish TRPM2 channel in the presence ... -

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Basic information

Entry
Database: PDB / ID: 6d73
TitleCryo-EM structure of the zebrafish TRPM2 channel in the presence of Ca2+
ComponentsTransient receptor potential cation channel, subfamily M
KeywordsTRANSPORT PROTEIN / warmth sensor / redox sensor / calcium-permeable ion channel / ion channel
Biological speciesDanio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsYin, Y. / Wu, M. / Borschel, W.F. / Lander, G.C. / 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: Nat Commun / Year: 2019
Title: Visualizing structural transitions of ligand-dependent gating of the TRPM2 channel.
Authors: Ying Yin / Mengyu Wu / Allen L Hsu / William F Borschel / Mario J Borgnia / Gabriel C Lander / Seok-Yong Lee /
Abstract: The transient receptor potential melastatin 2 (TRPM2) channel plays a key role in redox sensation in many cell types. Channel activation requires binding of both ADP-ribose (ADPR) and Ca. The ...The transient receptor potential melastatin 2 (TRPM2) channel plays a key role in redox sensation in many cell types. Channel activation requires binding of both ADP-ribose (ADPR) and Ca. The recently published TRPM2 structures from Danio rerio in the ligand-free and the ADPR/Ca-bound conditions represent the channel in closed and open states, which uncovered substantial tertiary and quaternary conformational rearrangements. However, it is unclear how these rearrangements are achieved within the tetrameric channel during channel gating. Here we report the cryo-electron microscopy structures of Danio rerio TRPM2 in the absence of ligands, in complex with Ca alone, and with both ADPR and Ca, resolved to ~4.3 Å, ~3.8 Å, and ~4.2 Å, respectively. In contrast to the published results, our studies capture ligand-bound TRPM2 structures in two-fold symmetric intermediate states, offering a glimpse of the structural transitions that bridge the closed and open conformations.
History
DepositionApr 23, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 15, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 31, 2019Group: Data collection / Structure summary / Category: struct / Item: _struct.title
Revision 1.2Nov 27, 2019Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / cell / 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] / _cell.Z_PDB / _pdbx_audit_support.country / _pdbx_audit_support.funding_organization
Revision 1.4Nov 6, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)670,5958
Polymers670,4354
Non-polymers1604
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Transient receptor potential cation channel, subfamily M


Mass: 167608.625 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: trpm2 / Production host: Homo sapiens (human)
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
Has protein modificationY

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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 cation channel subfamily M member 2 (TRPM2)
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Danio rerio (zebrafish)
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 supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 16 sec. / Electron dose: 63 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3039
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 64 / Used frames/image: 1-64

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2Leginon3.2image acquisitionAutomated data acquisition software
4CTFFIND4CTF correction
5RELION2.1CTF correction
8Coot0.8.8model fitting
10RELION2.1initial Euler assignment
11RELION2.1final Euler assignment
13RELION2.13D reconstruction
14PHENIX1.12-2829-000model refinement
15Coot0.8.8model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1791114
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93573 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 100 / Protocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00436716
ELECTRON MICROSCOPYf_angle_d0.68350298
ELECTRON MICROSCOPYf_dihedral_angle_d8.62421224
ELECTRON MICROSCOPYf_chiral_restr0.0415948
ELECTRON MICROSCOPYf_plane_restr0.0056374

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