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- PDB-6c9a: Cryo-EM structure of mouse TPC1 channel in the PtdIns(3,5)P2-boun... -

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Basic information

Entry
Database: PDB / ID: 6c9a
TitleCryo-EM structure of mouse TPC1 channel in the PtdIns(3,5)P2-bound state
ComponentsTwo pore calcium channel protein 1
KeywordsMEMBRANE PROTEIN / Ion channel
Function / homology
Function and homology information


voltage-gated channel activity / endolysosome / intracellularly phosphatidylinositol-3,5-bisphosphate-gated monatomic cation channel activity / NAADP-sensitive calcium-release channel activity / Stimuli-sensing channels / phosphatidylinositol-3,5-bisphosphate binding / syntaxin binding / ligand-gated sodium channel activity / voltage-gated sodium channel activity / monoatomic ion channel complex ...voltage-gated channel activity / endolysosome / intracellularly phosphatidylinositol-3,5-bisphosphate-gated monatomic cation channel activity / NAADP-sensitive calcium-release channel activity / Stimuli-sensing channels / phosphatidylinositol-3,5-bisphosphate binding / syntaxin binding / ligand-gated sodium channel activity / voltage-gated sodium channel activity / monoatomic ion channel complex / positive regulation of autophagy / sodium ion transmembrane transport / recycling endosome membrane / early endosome membrane / endosome membrane / endocytosis involved in viral entry into host cell / lysosomal membrane / protein homodimerization activity / membrane
Similarity search - Function
Two pore channel protein 1 / Voltage-dependent channel domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Chem-EUJ / Two pore calcium channel protein 1
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsShe, J. / Guo, J. / Chen, Q. / Bai, X. / Jiang, Y.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR)GM079179 United States
Welch FoundationI-1578 United States
Howard Hughes Medical Institute (HHMI) United States
Cancer Prevention and Research Institute of Texas (CPRIT) United States
Virginia Murchison Linthicum Scholar in Medical Research fund United States
CitationJournal: Nature / Year: 2018
Title: Structural insights into the voltage and phospholipid activation of the mammalian TPC1 channel.
Authors: Ji She / Jiangtao Guo / Qingfeng Chen / Weizhong Zeng / Youxing Jiang / Xiao-Chen Bai /
Abstract: The organellar two-pore channel (TPC) functions as a homodimer, in which each subunit contains two homologous Shaker-like six-transmembrane (6-TM)-domain repeats. TPCs belong to the voltage-gated ion ...The organellar two-pore channel (TPC) functions as a homodimer, in which each subunit contains two homologous Shaker-like six-transmembrane (6-TM)-domain repeats. TPCs belong to the voltage-gated ion channel superfamily and are ubiquitously expressed in animals and plants. Mammalian TPC1 and TPC2 are localized at the endolysosomal membrane, and have critical roles in regulating the physiological functions of these acidic organelles. Here we present electron cryo-microscopy structures of mouse TPC1 (MmTPC1)-a voltage-dependent, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P)-activated Na-selective channel-in both the apo closed state and ligand-bound open state. Combined with functional analysis, these structures provide comprehensive structural insights into the selectivity and gating mechanisms of mammalian TPC channels. The channel has a coin-slot-shaped ion pathway in the filter that defines the selectivity of mammalian TPCs. Only the voltage-sensing domain from the second 6-TM domain confers voltage dependence on MmTPC1. Endolysosome-specific PtdIns(3,5)P binds to the first 6-TM domain and activates the channel under conditions of depolarizing membrane potential. Structural comparisons between the apo and PtdIns(3,5)P-bound structures show the interplay between voltage and ligand in channel activation. These MmTPC1 structures reveal lipid binding and regulation in a 6-TM voltage-gated channel, which is of interest in light of the emerging recognition of the importance of phosphoinositide regulation of ion channels.
History
DepositionJan 25, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 4, 2018Provider: repository / Type: Initial release
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Revision 1.1Apr 18, 2018Group: Data collection / Database references / Category: citation
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Revision 1.2Apr 24, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
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Description: Carbohydrate remediation / Provider: repository / Type: Remediation
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Structure visualization

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Assembly

Deposited unit
B: Two pore calcium channel protein 1
A: Two pore calcium channel protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)193,6148
Polymers190,8302
Non-polymers2,7846
Water00
1


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

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Components

#1: Protein Two pore calcium channel protein 1 / Voltage-dependent calcium channel protein TPC1


Mass: 95414.758 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Tpcn1, Kiaa1169, Tpc1 / Production host: Homo sapiens (human) / References: UniProt: Q9EQJ0
#2: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#3: Chemical ChemComp-EUJ / (2R)-3-{[(S)-hydroxy{[(1S,2R,3R,4S,5S,6R)-2,4,6-trihydroxy-3,5-bis(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}propane-1,2-diyl dioctanoate


Mass: 746.566 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C25H49O19P3
#4: 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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
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
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: PtdIns(3,5)P2-bound mouse TPC1 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F / Plasmid: pEZT-BM
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 FIELD
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 82819 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00912490
ELECTRON MICROSCOPYf_angle_d1.00416926
ELECTRON MICROSCOPYf_dihedral_angle_d15.0687406
ELECTRON MICROSCOPYf_chiral_restr0.0571896
ELECTRON MICROSCOPYf_plane_restr0.0072074

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