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- PDB-8srf: Cryo-EM structure of TRPM2 chanzyme in the presence of Magnesium,... -

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Basic information

Entry
Database: PDB / ID: 8srf
TitleCryo-EM structure of TRPM2 chanzyme in the presence of Magnesium, ADP-ribose, Adenosine monophosphate, and Ribose-5-phosphate, closed state
ComponentsTRPM2 chanzyme
KeywordsMEMBRANE PROTEIN / TRPM2 Chanzyme / Channel-enzyme
Function / homology
Function and homology information


endomembrane system / calcium channel activity / plasma membrane
Similarity search - Function
TRPM, SLOG domain / : / SLOG in TRPM / TRPM2-like domain / Nudix hydrolase domain profile. / NUDIX hydrolase domain / Voltage-dependent channel domain superfamily / NUDIX hydrolase-like domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
ADENOSINE MONOPHOSPHATE / ADENOSINE-5-DIPHOSPHORIBOSE / CHOLESTEROL / 5-O-phosphono-beta-D-ribofuranose / Nudt9 protein
Similarity search - Component
Biological speciesSalpingoeca rosetta (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.52 Å
AuthorsHuang, Y. / Kumar, S. / Lu, W. / Du, J.
Funding support United States, 4items
OrganizationGrant numberCountry
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
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS111031 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Coupling enzymatic activity and gating in an ancient TRPM chanzyme and its molecular evolution.
Authors: Yihe Huang / Sushant Kumar / Junuk Lee / Wei Lü / Juan Du /
Abstract: Channel enzymes represent a class of ion channels with enzymatic activity directly or indirectly linked to their channel function. We investigated a TRPM2 chanzyme from choanoflagellates that ...Channel enzymes represent a class of ion channels with enzymatic activity directly or indirectly linked to their channel function. We investigated a TRPM2 chanzyme from choanoflagellates that integrates two seemingly incompatible functions into a single peptide: a channel module activated by ADP-ribose with high open probability and an enzyme module (NUDT9-H domain) consuming ADP-ribose at a remarkably slow rate. Using time-resolved cryogenic-electron microscopy, we captured a complete series of structural snapshots of gating and catalytic cycles, revealing the coupling mechanism between channel gating and enzymatic activity. The slow kinetics of the NUDT9-H enzyme module confers a self-regulatory mechanism: ADPR binding triggers NUDT9-H tetramerization, promoting channel opening, while subsequent hydrolysis reduces local ADPR, inducing channel closure. We further demonstrated how the NUDT9-H domain has evolved from a structurally semi-independent ADP-ribose hydrolase module in early species to a fully integrated component of a gating ring essential for channel activation in advanced species.
History
DepositionMay 5, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 15, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.2Oct 23, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update
Revision 1.3Oct 30, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.4Nov 13, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: TRPM2 chanzyme
B: TRPM2 chanzyme
C: TRPM2 chanzyme
D: TRPM2 chanzyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)682,70749
Polymers673,0104
Non-polymers9,69745
Water724
1


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

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Components

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Protein / Sugars , 2 types, 8 molecules ABCD

#1: Protein
TRPM2 chanzyme / Nudt9 protein


Mass: 168252.484 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salpingoeca rosetta (eukaryote) / Gene: PTSG_05449 / Production host: Homo sapiens (human) / References: UniProt: F2UB89
#2: Sugar
ChemComp-RP5 / 5-O-phosphono-beta-D-ribofuranose / [(2R,3S,4S,5R)-3,4,5-TRIHYDROXYTETRAHYDROFURAN-2-YL]METHYL DIHYDROGEN PHOSPHATE / 5-O-phosphono-beta-D-ribose / 5-O-phosphono-D-ribose / 5-O-phosphono-ribose


Type: D-saccharide, beta linking / Mass: 230.110 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C5H11O8P / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
b-D-Ribf5PO3IUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0

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Non-polymers , 5 types, 45 molecules

#3: Chemical
ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM
#4: Chemical
ChemComp-APR / ADENOSINE-5-DIPHOSPHORIBOSE


Mass: 559.316 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C15H23N5O14P2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 21 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
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: TRPM2 chanzyme incubated with Magnesium and ADP-ribose for 10s; ADP-ribose partially hydrolyzed; intact ADP-ribose in MHR1/2 domain; Adenosine monophosphate and Ribose-5-phosphate in NUDT9-H domain; closed state.
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Salpingoeca rosetta (eukaryote)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenConc.: 8.5 mg/ml / Embedding 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 FIELD / Nominal defocus max: 1900 nm / Nominal defocus min: 900 nm
Image recordingAverage exposure time: 0.02 sec. / Electron dose: 49 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM softwareName: PHENIX / Version: 1.20.1_4487 / Category: model refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1761 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01643772
ELECTRON MICROSCOPYf_angle_d1.90959548
ELECTRON MICROSCOPYf_dihedral_angle_d5.6296032
ELECTRON MICROSCOPYf_chiral_restr0.0936768
ELECTRON MICROSCOPYf_plane_restr0.0127556

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