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- PDB-6cx0: Structure of AtTPC1 D376A -

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Basic information

Entry
Database: PDB / ID: 6cx0
TitleStructure of AtTPC1 D376A
ComponentsTwo pore calcium channel protein 1
Keywordsmembrane protein/inhibitor / Ion channel / Two-pore channel / TPC1 / resting-state / closed / inactive / MEMBRANE PROTEIN / membrane protein-inhibitor complex
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 / calcium-mediated signaling / calcium ion transport ...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 / 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 / EF-hand, calcium binding motif / EF-hand calcium-binding domain profile. / Ion transport domain / Ion transport protein / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
Chem-FJ7 / Two pore calcium channel protein 1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.501 Å
AuthorsKintzer, A.F. / Stroud, R.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM24485 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Structural basis for activation of voltage sensor domains in an ion channel TPC1.
Authors: Alexander F Kintzer / Evan M Green / Pawel K Dominik / Michael Bridges / Jean-Paul Armache / Dawid Deneka / Sangwoo S Kim / Wayne Hubbell / Anthony A Kossiakoff / Yifan Cheng / Robert M Stroud /
Abstract: Voltage-sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids ...Voltage-sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured an example of a resting-state VSD in an intact ion channel. To generate an activated-state VSD in the same channel we removed the luminal inhibitory Ca-binding site (Ca), which shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca to a cytoplasmic site (Ca). An X-ray structure with Ca removed and a near-atomic resolution cryo-EM structure with Ca removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage-dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel's requirement of cytoplasmic Ca ions for activation.
History
DepositionApr 2, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 19, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 3, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Oct 4, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_symmetry / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)85,0257
Polymers84,3101
Non-polymers7156
Water0
1
A: Two pore calcium channel protein 1
hetero molecules

A: Two pore calcium channel protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)170,05014
Polymers168,6202
Non-polymers1,43012
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_555x,-y,-z1
Buried area8700 Å2
ΔGint-191 kcal/mol
Surface area65630 Å2
MethodPISA
Unit cell
Length a, b, c (Å)88.470, 154.420, 218.450
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221

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Components

#1: Protein Two pore calcium channel protein 1 / 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: 84310.234 Da / Num. of mol.: 1 / Mutation: D376A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: TPC1, CCH1, FOU2, At4g03560, F9H3.19, T5L23.5 / Plasmid: p423Gal1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): DSY5 / Variant (production host): -HIS / References: UniProt: Q94KI8
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Formula: Ca
#3: Chemical ChemComp-FJ7 / (1S,3R)-1-(3-{[4-(2-fluorophenyl)piperazin-1-yl]methyl}-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-beta-carboline-3-carboxylic acid


Mass: 514.591 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C30H31FN4O3

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.42 Å3/Da / Density % sol: 72.2 %
Crystal growTemperature: 292 K / Method: vapor diffusion, sitting drop / pH: 9.3
Details: Sitting drop 2microliter drops crystchem 0.1 M glycine, pH 9.3, 50 mM potassium chloride, 1 mM calcium chloride, 35% PEG300

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Feb 21, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.5→30 Å / Num. obs: 19231 / % possible obs: 99.7 % / Redundancy: 13.6 % / Rrim(I) all: 0.239 / Net I/σ(I): 8.08
Reflection shellResolution: 3.5→4 Å / Num. unique obs: 6273 / % possible all: 99.8

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Processing

Software
NameVersionClassification
PHENIX(1.11.1_2575: ???)refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5DQQ

5dqq


Resolution: 3.501→41 Å / SU ML: 0.5 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 51.07 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3519 444 4.96 %
Rwork0.3184 --
obs0.3199 8953 46.46 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.501→41 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5049 0 43 0 5092
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0035218
X-RAY DIFFRACTIONf_angle_d0.6877098
X-RAY DIFFRACTIONf_dihedral_angle_d16.8733005
X-RAY DIFFRACTIONf_chiral_restr0.04810
X-RAY DIFFRACTIONf_plane_restr0.005867
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.5009-4.00710.4289230.3796460X-RAY DIFFRACTION8
4.0071-5.0470.36451120.32442270X-RAY DIFFRACTION38
5.047-41.0030.34723090.31525779X-RAY DIFFRACTION93

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