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- EMDB-31587: Structure of AtTPC1 D240A/D454A/E528A mutant with 1 mM Ca2+ -

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Basic information

Entry
Database: EMDB / ID: EMD-31587
TitleStructure of AtTPC1 D240A/D454A/E528A mutant with 1 mM Ca2+
Map dataStructure of AtTPC1 D240A/D454A/E528A mutant with 1 mM Ca2
Sample
  • Complex: AtTPC1 D240A/D454A/E528A mutant homodimer
    • Protein or peptide: Two pore calcium channel protein 1,GFP
  • Ligand: CALCIUM ION
Keywordsnon-selective cation channel / dimer / vacuole / TRANSPORT PROTEIN
Function / homology
Function and homology information


regulation of jasmonic acid biosynthetic process / seed germination / regulation of stomatal movement / plant-type vacuole / vacuole / vacuolar membrane / monoatomic ion channel complex / voltage-gated calcium channel activity / bioluminescence / generation of precursor metabolites and energy ...regulation of jasmonic acid biosynthetic process / seed germination / regulation of stomatal movement / plant-type vacuole / vacuole / vacuolar membrane / monoatomic ion channel complex / voltage-gated calcium channel activity / bioluminescence / generation of precursor metabolites and energy / calcium-mediated signaling / calcium ion transport / calcium ion binding / Golgi apparatus / identical protein binding / plasma membrane / cytosol
Similarity search - Function
Two pore calcium channel protein 1, plant / Voltage-dependent channel domain superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / EF-hand, calcium binding motif / EF-hand calcium-binding domain profile. / EF-hand domain / Ion transport domain ...Two pore calcium channel protein 1, plant / Voltage-dependent channel domain superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / EF-hand, calcium binding motif / EF-hand calcium-binding domain profile. / EF-hand domain / Ion transport domain / Ion transport protein / EF-hand domain pair
Similarity search - Domain/homology
GFP / Two pore calcium channel protein 1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress) / Human respiratory syncytial virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsYe F / Xu L
Funding support China, 3 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2020YFA0908501 China
Ministry of Science and Technology (MoST, China)2018YFA0508100 China
National Natural Science Foundation of China (NSFC)31870724 China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Voltage-gating and cytosolic Ca activation mechanisms of two-pore channel AtTPC1.
Authors: Fan Ye / Lingyi Xu / Xiaoxiao Li / Weizhong Zeng / Ninghai Gan / Cheng Zhao / Wei Yang / Youxing Jiang / Jiangtao Guo /
Abstract: two-pore channel AtTPC1 is a voltage-gated, Ca-modulated, nonselective cation channel that is localized in the vacuolar membrane and responsible for generating slow vacuolar (SV) current. Under ... two-pore channel AtTPC1 is a voltage-gated, Ca-modulated, nonselective cation channel that is localized in the vacuolar membrane and responsible for generating slow vacuolar (SV) current. Under depolarizing membrane potential, cytosolic Ca activates AtTPC1 by binding at the EF-hand domain, whereas luminal Ca inhibits the channel by stabilizing the voltage-sensing domain II (VSDII) in the resting state. Here, we present 2.8 to 3.3 Å cryoelectron microscopy (cryo-EM) structures of AtTPC1 in two conformations, one in closed conformation with unbound EF-hand domain and resting VSDII and the other in a partially open conformation with Ca-bound EF-hand domain and activated VSDII. Structural comparison between the two different conformations allows us to elucidate the structural mechanisms of voltage gating, cytosolic Ca activation, and their coupling in AtTPC1. This study also provides structural insight into the general voltage-gating mechanism among voltage-gated ion channels.
History
DepositionJul 29, 2021-
Header (metadata) releaseDec 1, 2021-
Map releaseDec 1, 2021-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
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  • Surface view colored by cylindrical radius
  • Surface level: 0.015
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  • Surface view with fitted model
  • Atomic models: PDB-7fhn
  • Surface level: 0.015
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31587.png

Downloads & links

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Map

FileDownload / File: emd_31587.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of AtTPC1 D240A/D454A/E528A mutant with 1 mM Ca2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 240 pix.
= 243.36 Å		1.01 Å/pix.
x 240 pix.
= 243.36 Å		1.01 Å/pix.
x 240 pix.
= 243.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.014 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.042399272 - 0.08243881
Average (Standard dev.)0.00015544555 (±0.0019147375)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 243.36002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0141.0141.014
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z243.360243.360243.360
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0420.0820.000

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Supplemental data

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Sample components

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Entire : AtTPC1 D240A/D454A/E528A mutant homodimer

EntireName: AtTPC1 D240A/D454A/E528A mutant homodimer
Components
  • Complex: AtTPC1 D240A/D454A/E528A mutant homodimer
    • Protein or peptide: Two pore calcium channel protein 1,GFP
  • Ligand: CALCIUM ION

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Supramolecule #1: AtTPC1 D240A/D454A/E528A mutant homodimer

SupramoleculeName: AtTPC1 D240A/D454A/E528A mutant homodimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Arabidopsis thaliana (thale cress)

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Macromolecule #1: Two pore calcium channel protein 1,GFP

MacromoleculeName: Two pore calcium channel protein 1,GFP / type: protein_or_peptide / ID: 1
Details: The fusion protein of AtTPC1 (UNP residues 1-733), LINKER and EGFP (UNP residues 2-244).
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Human respiratory syncytial virus
Molecular weightTheoretical: 114.49825 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MEDPLIGRDS LGGGGTDRVR RSEAITHGTP FQKAAALVDL AEDGIGLPVE ILDQSSFGES ARYYFIFTRL DLIWSLNYFA LLFLNFFEQ PLWCEKNPKP SCKDRDYYYL GELPYLTNAE SIIYEVITLA ILLVHTFFPI SYEGSRIFWT SRLNLVKVAC V VILFVDVL ...String:
MEDPLIGRDS LGGGGTDRVR RSEAITHGTP FQKAAALVDL AEDGIGLPVE ILDQSSFGES ARYYFIFTRL DLIWSLNYFA LLFLNFFEQ PLWCEKNPKP SCKDRDYYYL GELPYLTNAE SIIYEVITLA ILLVHTFFPI SYEGSRIFWT SRLNLVKVAC V VILFVDVL VDFLYLSPLA FDFLPFRIAP YVRVIIFILS IRELRDTLVL LSGMLGTYLN ILALWMLFLL FASWIAFVMF EA TQQGLTV FTSYGATLYQ MFILFTTSNN PDVWIPAYKS SRWSSVFFVL YVLIGVYFVT NLILAVVYDS FKEQLAKQVS GMD QMKRRM LEKAFGLIDS DKNGEIDKNQ CIKLFEQLTN YRTLPKISKE EFGLIFDELD DTRDFKINKD EFADLCQAIA LRFQ KEEVP SLFEHFPQIY HSALSQQLRA FVRSPNFGYA ISFILIINFI AVVVETTLAI EESSAQKPWQ VAEFVFGWIY VLEMA LKIY TYGFENYWRE GANRFDFLVT WVIVIGETAT FITPDENTFF SNGAWIRYLL LARMLRLIRL LMNVQRYRAF IATFIT LIP SLMPYLGTIF CVLCIYCSIG VQVFGGLVNA GNKKLFETEL AEDDYLLFNF NDYPNGMVTL FNLLVMGNWQ VWMESYK DL TGTWWSITYF VSFYVITILL LLNLVVAFVL EAFFTELDLE EEEKCQGQDS QEKRNRRRSA GSKSRSQRVD TLLHHMLG D ELSKPECSTS DTSTAGLVPR GSAAAAVSKG EELFTGVVPI LVELDGDVNG HKFSVSGEGE GDATYGKLTL KFICTTGKL PVPWPTLVTT LTYGVQCFSR YPDHMKQHDF FKSAMPEGYV QERTIFFKDD GNYKTRAEVK FEGDTLVNRI ELKGIDFKED GNILGHKLE YNYNSHNVYI MADKQKNGIK VNFKIRHNIE DGSVQLADHY QQNTPIGDGP VLLPDNHYLS TQSKLSKDPN E KRDHMVLL EFVTAAGITL GMDELYKSGL RSHHHHHHHH

UniProtKB: Two pore calcium channel protein 1, GFP

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 64.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 44139
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER

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