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- EMDB-25827: AtTPC1 D454N with 1 mM EDTA state I -

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Basic information

Entry
Database: EMDB / ID: EMD-25827
TitleAtTPC1 D454N with 1 mM EDTA state I
Map dataVolume from non-uniform refinement, post-processed with Deepemhancer (inputs are the two provided half maps)
Sample
  • Complex: AtTPC1 D454N with 1 mM EDTA state I
    • Protein or peptide: Two pore calcium channel protein 1
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: 2.7 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 31, 2021-
Header (metadata) releaseFeb 2, 2022-
Map releaseFeb 2, 2022-
UpdateMar 9, 2022-
Current statusMar 9, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0905
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0905
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7tdf
  • Surface level: 0.0905
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25827.png

Downloads & links

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Map

FileDownload / File: emd_25827.map.gz / Format: CCP4 / Size: 219.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVolume from non-uniform refinement, post-processed with Deepemhancer (inputs are the two provided half maps)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 386 pix.
= 322.31 Å		0.84 Å/pix.
x 386 pix.
= 322.31 Å		0.84 Å/pix.
x 386 pix.
= 322.31 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0905 / Movie #1: 0.0905
Minimum - Maximum-0.021287838 - 1.8625423
Average (Standard dev.)0.00068822084 (±0.016633773)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS386386386
D min/max/mean-0.0211.8630.001

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Supplemental data

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Additional map: #1

Fileemd_25827_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 from non-uniform refinement

Fileemd_25827_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_25827_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 : AtTPC1 D454N with 1 mM EDTA state I

EntireName: AtTPC1 D454N with 1 mM EDTA state I
Components
  • Complex: AtTPC1 D454N with 1 mM EDTA state I
    • Protein or peptide: Two pore calcium channel protein 1

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Supramolecule #1: AtTPC1 D454N with 1 mM EDTA state I

SupramoleculeName: AtTPC1 D454N with 1 mM EDTA state I / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
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: 84.939844 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 EESSAQKPWQ 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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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
Details: 50 mM Tris, 200 mM NaCl, 0.06% glycodiosgenin, 1 mM EDTA
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

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Electron microscopy

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

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

25798

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 129676
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-7tdf:
AtTPC1 D454N with 1 mM EDTA state I

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