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- EMDB-0479: Cryo-EM structure of human TPC2 channel in the ligand-bound close... -

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Basic information

Entry
Database: EMDB / ID: EMD-0479
TitleCryo-EM structure of human TPC2 channel in the ligand-bound closed state
Map dataHuman TPC2 channel in the ligand-bound closed state
Sample
  • Complex: The complex of two-pore channel 2 with PI(3,5)P2
    • Protein or peptide: Two pore calcium channel protein 2
  • Ligand: (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
Function / homology
Function and homology information


endosome to lysosome transport of low-density lipoprotein particle / negative regulation of developmental pigmentation / intracellular pH reduction / intracellularly phosphatidylinositol-3,5-bisphosphate-gated monatomic cation channel activity / ligand-gated sodium channel activity / NAADP-sensitive calcium-release channel activity / melanosome membrane / regulation of exocytosis / response to vitamin D / endolysosome membrane ...endosome to lysosome transport of low-density lipoprotein particle / negative regulation of developmental pigmentation / intracellular pH reduction / intracellularly phosphatidylinositol-3,5-bisphosphate-gated monatomic cation channel activity / ligand-gated sodium channel activity / NAADP-sensitive calcium-release channel activity / melanosome membrane / regulation of exocytosis / response to vitamin D / endolysosome membrane / phosphatidylinositol-3,5-bisphosphate binding / monoatomic ion channel complex / lysosome organization / sodium ion transmembrane transport / smooth muscle contraction / voltage-gated calcium channel activity / release of sequestered calcium ion into cytosol / monoatomic ion transmembrane transport / regulation of autophagy / calcium-mediated signaling / endocytosis involved in viral entry into host cell / calcium channel activity / intracellular calcium ion homeostasis / Stimuli-sensing channels / late endosome membrane / receptor-mediated endocytosis of virus by host cell / lysosome / endosome membrane / lysosomal membrane / protein kinase binding / identical protein binding / cytosol
Similarity search - Function
Two pore channel protein 2 / Voltage-dependent channel domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Two pore channel protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsShe J / Zeng W / Guo J / Chen Q / Bai X / Jiang Y
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM079179 United States
Welch FoundationGrant I-1578
Howard Hughes Medical Institute (HHMI) United States
Cancer Prevention and Research Institute of Texas (CPRIT) United States
CitationJournal: Elife / Year: 2019
Title: Structural mechanisms of phospholipid activation of the human TPC2 channel.
Authors: Ji She / Weizhong Zeng / Jiangtao Guo / Qingfeng Chen / Xiao-Chen Bai / Youxing Jiang /
Abstract: Mammalian two-pore channels (TPCs) regulate the physiological functions of the endolysosome. Here we present cryo-EM structures of human TPC2 (HsTPC2), a phosphatidylinositol 3,5-bisphosphate (PI(3,5) ...Mammalian two-pore channels (TPCs) regulate the physiological functions of the endolysosome. Here we present cryo-EM structures of human TPC2 (HsTPC2), a phosphatidylinositol 3,5-bisphosphate (PI(3,5)P)-activated, Na selective channel, in the ligand-bound and apo states. The apo structure captures the closed conformation, while the ligand-bound form features the channel in both open and closed conformations. Combined with functional analysis, these structures provide insights into the mechanism of PI(3,5)P-regulated gating of TPC2, which is distinct from that of TPC1. Specifically, the endolysosome-specific PI(3,5)P binds at the first 6-TM and activates the channel - independently of the membrane potential - by inducing a structural change at the pore-lining inner helix (IS6), which forms a continuous helix in the open state but breaks into two segments at Gly317 in the closed state. Additionally, structural comparison to the voltage-dependent TPC1 structure allowed us to identify Ile551 as being responsible for the loss of voltage dependence in TPC2.
History
DepositionJan 18, 2019-
Header (metadata) releaseMar 6, 2019-
Map releaseMar 27, 2019-
UpdateNov 20, 2019-
Current statusNov 20, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nq2
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0479.png

Downloads & links

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Map

FileDownload / File: emd_0479.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman TPC2 channel in the ligand-bound closed state
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.13506703 - 0.23433577
Average (Standard dev.)-0.00001025510 (±0.008334627)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 256.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z256.800256.800256.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-383-383-383
NX/NY/NZ768768768
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.1350.234-0.000

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Supplemental data

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Sample components

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Entire : The complex of two-pore channel 2 with PI(3,5)P2

EntireName: The complex of two-pore channel 2 with PI(3,5)P2
Components
  • Complex: The complex of two-pore channel 2 with PI(3,5)P2
    • Protein or peptide: Two pore calcium channel protein 2
  • Ligand: (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

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Supramolecule #1: The complex of two-pore channel 2 with PI(3,5)P2

SupramoleculeName: The complex of two-pore channel 2 with PI(3,5)P2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Two pore calcium channel protein 2

MacromoleculeName: Two pore calcium channel protein 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 85.671828 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GSLEMAEPQA ESEPAAGGAR GGGGDWPAGL TTYRSIQVGP GAAARWDLCI DQAVVFIEDA IQYRSINHRV DASSMWLYRR YYSNVCQRT LSFTIFLILF LAFIETPSSL TSTADVRYRA APWEPPCGLT ESVEVLCLLV FAADLSVKGY LFGWAHFQKN L WLLGYLVV ...String:
GSLEMAEPQA ESEPAAGGAR GGGGDWPAGL TTYRSIQVGP GAAARWDLCI DQAVVFIEDA IQYRSINHRV DASSMWLYRR YYSNVCQRT LSFTIFLILF LAFIETPSSL TSTADVRYRA APWEPPCGLT ESVEVLCLLV FAADLSVKGY LFGWAHFQKN L WLLGYLVV LVVSLVDWTV SLSLVCHEPL RIRRLLRPFF LLQNSSMMKK TLKCIRWSLP EMASVGLLLA IHLCLFTMFG ML LFAGGKQ DDGQDRERLT YFQNLPESLT SLLVLLTTAN NPDVMIPAYS KNRAYAIFFI VFTVIGSLFL MNLLTAIIYS QFR GYLMKS LQTSLFRRRL GTRAAFEVLS SMVGEGGAFP QAVGVKPQNL LQVLQKVQLD SSHKQAMMEK VRSYGSVLLS AEEF QKLFN ELDRSVVKEH PPRPEYQSPF LQSAQFLFGH YYFDYLGNLI ALANLVSICV FLVLDADVLP AERDDFILGI LNCVF IVYY LLEMLLKVFA LGLRGYLSYP SNVFDGLLTV VLLVLEISTL AVYRLPHPGW RPEMVGLLSL WDMTRMLNML IVFRFL RII PSMKPMAVVA STVLGLVQNM RAFGGILVVV YYVFAIIGIN LFRGVIVALP GNSSLAPANG SAPCGSFEQL EYWANNF DD FAAALVTLWN LMVVNNWQVF LDAYRRYSGP WSKIYFVLWW LVSSVIWVNL FLALILENFL HKWDPRSHLQ PLAGTPEA T YQMTVELLFR DILEEPEEDE LTERLSQHPH LWLCR

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Macromolecule #2: (2R)-3-{[(S)-hydroxy{[(1S,2R,3R,4S,5S,6R)-2,4,6-trihydroxy-3,5-bi...

MacromoleculeName: (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
type: ligand / ID: 2 / Number of copies: 2 / Formula: EUJ
Molecular weightTheoretical: 746.566 Da
Chemical component information

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

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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

BufferpH: 8
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 56548

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