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- PDB-7mbt: Cryo-EM structure of zebrafish TRPM5 E337A mutant in the presence... -

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Basic information

Entry
Database: PDB / ID: 7mbt
TitleCryo-EM structure of zebrafish TRPM5 E337A mutant in the presence of 5 mM calcium (low calcium occupancy in the transmembrane domain)
ComponentsTransient receptor potential melastatin 5
KeywordsTRANSPORT PROTEIN / Ion channel / TRP channel
Function / homology
Function and homology information


ligand-gated calcium channel activity / calcium-activated cation channel activity / calcium ion transmembrane transport / membrane
Similarity search - Function
Transient receptor potential channel, canonical / TRPM, SLOG domain / SLOG in TRPM / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
(25R)-14beta,17beta-spirost-5-en-3beta-ol / Chem-YUY / Transient receptor potential melastatin 5
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM
AuthorsRuan, Z. / Lu, W. / Du, J. / Haley, E.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R56HL144929 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS111031 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL153219 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS112363 United States
American Heart Association20POST35120556 United States
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: Structures of the TRPM5 channel elucidate mechanisms of activation and inhibition.
Authors: Zheng Ruan / Emery Haley / Ian J Orozco / Mark Sabat / Richard Myers / Rebecca Roth / Juan Du / Wei Lü /
Abstract: The Ca-activated TRPM5 channel plays essential roles in taste perception and insulin secretion. However, the mechanism by which Ca regulates TRPM5 activity remains elusive. We report cryo-EM ...The Ca-activated TRPM5 channel plays essential roles in taste perception and insulin secretion. However, the mechanism by which Ca regulates TRPM5 activity remains elusive. We report cryo-EM structures of the zebrafish TRPM5 in an apo closed state, a Ca-bound open state, and an antagonist-bound inhibited state. We define two novel ligand binding sites: a Ca site (Ca) in the intracellular domain and an antagonist site in the transmembrane domain (TMD). The Ca site is unique to TRPM5 and has two roles: modulating the voltage dependence and promoting Ca binding to the Ca site, which is conserved throughout TRPM channels. Conformational changes initialized from both Ca sites cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the transmembrane domain in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents.
History
DepositionApr 1, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 7, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 28, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Transient receptor potential melastatin 5
B: Transient receptor potential melastatin 5
C: Transient receptor potential melastatin 5
D: Transient receptor potential melastatin 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)536,87116
Polymers530,9644
Non-polymers5,90712
Water0
1


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

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Components

#1: Protein
Transient receptor potential melastatin 5


Mass: 132740.953 Da / Num. of mol.: 4 / Mutation: E337A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: trpm5, TRPM5 / Production host: Homo sapiens (human) / References: UniProt: S5UH55
#2: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Chemical
ChemComp-YUY / (2R)-2-(hydroxymethyl)-4-{[(25R)-10alpha,14beta,17beta-spirost-5-en-3beta-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside


Mass: 841.033 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C44H72O15 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-YUV / (25R)-14beta,17beta-spirost-5-en-3beta-ol / Diosgenin


Mass: 414.621 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C27H42O3 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: TRPM5 channel E337A mutant / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Danio rerio (zebrafish)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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
Image recordingElectron dose: 47 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19rc3_4028: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionNum. of particles: 208000 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00331492
ELECTRON MICROSCOPYf_angle_d0.48443024
ELECTRON MICROSCOPYf_dihedral_angle_d6.3244588
ELECTRON MICROSCOPYf_chiral_restr0.0375104
ELECTRON MICROSCOPYf_plane_restr0.0055268

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