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- EMDB-25798: AtTPC1 D454N with 1 mM Ca2+ -

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Basic information

Entry
Database: EMDB / ID: EMD-25798
TitleAtTPC1 D454N with 1 mM Ca2+
Map dataNon-uniform refinement, post-processed with Deepemhancer. Inputs are the two provided half maps, high resolution protocol.
Sample
  • Complex: Two-pore channel 1 dimer
    • Protein or peptide: Two pore calcium channel protein 1
  • Ligand: CALCIUM IONCalcium
  • Ligand: SODIUM IONSodium
  • Ligand: water
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. / EF-hand domain / Ion transport domain / Ion transport protein / EF-hand domain pair
Similarity search - Domain/homology
Two pore calcium channel protein 1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsDickinson MS / Stroud RM
Funding support United States, 1 items
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: 2022
Title: Molecular basis of multistep voltage activation in plant two-pore channel 1.
Authors: Miles Sasha Dickinson / Jinping Lu / Meghna Gupta / Irene Marten / Rainer Hedrich / Robert M Stroud /
Abstract: Voltage-gated ion channels confer excitability to biological membranes, initiating and propagating electrical signals across large distances on short timescales. Membrane excitation requires channels ...Voltage-gated ion channels confer excitability to biological membranes, initiating and propagating electrical signals across large distances on short timescales. Membrane excitation requires channels that respond to changes in electric field and couple the transmembrane voltage to gating of a central pore. To address the mechanism of this process in a voltage-gated ion channel, we determined structures of the plant two-pore channel 1 at different stages along its activation coordinate. These high-resolution structures of activation intermediates, when compared with the resting-state structure, portray a mechanism in which the voltage-sensing domain undergoes dilation and in-membrane plane rotation about the gating charge-bearing helix, followed by charge translocation across the charge transfer seal. These structures, in concert with patch-clamp electrophysiology, show that residues in the pore mouth sense inhibitory Ca and are allosterically coupled to the voltage sensor. These conformational changes provide insight into the mechanism of voltage-sensor domain activation in which activation occurs vectorially over a series of elementary steps.
History
DepositionDec 22, 2021-
Header (metadata) releaseFeb 2, 2022-
Map releaseFeb 2, 2022-
UpdateMar 16, 2022-
Current statusMar 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.109
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.109
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7tbg
  • Surface level: 0.109
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25798.png

Downloads & links

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Map

FileDownload / File: emd_25798.map.gz / Format: CCP4 / Size: 219.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNon-uniform refinement, post-processed with Deepemhancer. Inputs are the two provided half maps, high resolution protocol.
Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 0.109 / Movie #1: 0.109
Minimum - Maximum-0.026206804 - 1.8947906
Average (Standard dev.)0.00075819454 (±0.017054493)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions386386386
Spacing386386386
CellA=B=C: 322.31 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8350.8350.835
M x/y/z386386386
origin x/y/z0.0000.0000.000
length x/y/z322.310322.310322.310
α/β/γ90.00090.00090.000
start NX/NY/NZ154162115
NX/NY/NZ9384171
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS386386386
D min/max/mean-0.0261.8950.001

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Supplemental data

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Half map: Half map 1 from non-uniform refinement

Fileemd_25798_half_map_1.map
AnnotationHalf map 1 from non-uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 from non-uniform refinement

Fileemd_25798_half_map_2.map
AnnotationHalf map 2 from non-uniform refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Two-pore channel 1 dimer

EntireName: Two-pore channel 1 dimer
Components
  • Complex: Two-pore channel 1 dimer
    • Protein or peptide: Two pore calcium channel protein 1
  • Ligand: CALCIUM IONCalcium
  • Ligand: SODIUM IONSodium
  • Ligand: water

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Supramolecule #1: Two-pore channel 1 dimer

SupramoleculeName: Two-pore channel 1 dimer / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 168 KDa

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Macromolecule #1: Two pore calcium channel protein 1

MacromoleculeName: Two pore calcium channel protein 1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 84.956836 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
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 ED TQQGLTV FTSYGATLYQ MFILFTTSNN PDVWIPAYKS SRWSSVFFVL YVLIGVYFVT NLILAVVYDS FKEQLAKQVS GMD QMKRRM LEKAFGLIDS DKNGEIDKNQ CIKLFEQLTN YRTLPKISKE EFGLIFDELD DTRDFKINKD EFADLCQAIA LRFQ KEEVP SLFEHFPQIY HSALSQQLRA FVRSPNFGYA ISFILIINFI AVVVETTLNI EESSAQKNWQ VAEFVFGWIY VLEMA LKIY TYGFENYWRE GANRFDFLVT WVIVIGETAT FITPDENTFF SNGEWIRYLL LARMLRLIRL LMNVQRYRAF IATFIT LIP SLMPYLGTIF CVLCIYCSIG VQVFGGLVNA GNKKLFETEL AEDDYLLFNF NDYPNGMVTL FNLLVMGNWQ VWMESYK DL TGTWWSITYF VSFYVITILL LLNLVVAFVL EAFFTELDLE EEEKCQGQDS QEKRNRRRSA GSKSRSQRVD TLLHHMLG D ELSKPECSTS DT

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 3 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 20 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 280 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 66.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Stochastic gradient descent
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 140438
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7tbg:
AtTPC1 D454N with 1 mM Ca2+

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