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- PDB-7fho: Structure of AtTPC1 D240A/D454A/E528A mutant with 50 mM Ca2+ -

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Basic information

Entry
Database: PDB / ID: 7fho
TitleStructure of AtTPC1 D240A/D454A/E528A mutant with 50 mM Ca2+
ComponentsTwo pore calcium channel protein 1,GFP
KeywordsTRANSPORT PROTEIN / non-selective cation channel / dimer / vacuole
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
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsYe, F. / Xu, L. / Li, X. / Jiang, Y. / Guo, J.
Funding support China, 3items
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, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 1, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_DOI ..._citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Two pore calcium channel protein 1,GFP
C: Two pore calcium channel protein 1,GFP
hetero molecules


Theoretical massNumber of molelcules
Total (without water)229,2378
Polymers228,9972
Non-polymers2406
Water00
1


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

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Components

#1: Protein Two pore calcium channel protein 1,GFP / Calcium channel protein 1 / AtCCH1 / Fatty acid oxygenation up-regulated protein 2 / Voltage- ...Calcium channel protein 1 / AtCCH1 / Fatty acid oxygenation up-regulated protein 2 / Voltage-dependent calcium channel protein TPC1 / AtTPC1


Mass: 114498.250 Da / Num. of mol.: 2 / Mutation: D240A,D454A,E528A
Source method: isolated from a genetically manipulated source
Details: The fusion protein of AtTPC1 (UNP residues 1-733), LINKER and GFP (UNP residues 2-239)
Source: (gene. exp.) Arabidopsis thaliana (thale cress), (gene. exp.) Human respiratory syncytial virus
Gene: TPC1, CCH1, FOU2, At4g03560, F9H3.19, T5L23.5 / Production host: Homo sapiens (human) / References: UniProt: Q94KI8, UniProt: A0A5P9VSM6
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca / 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: AtTPC1 D240A/D454A/E528A mutant homodimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
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: OTHER
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 64 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 205220 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00210934
ELECTRON MICROSCOPYf_angle_d0.50914876
ELECTRON MICROSCOPYf_dihedral_angle_d3.6171406
ELECTRON MICROSCOPYf_chiral_restr0.0361686
ELECTRON MICROSCOPYf_plane_restr0.0041832

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