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- EMDB-8960: Structure of AtTPC1(DDE) in state 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-8960
TitleStructure of AtTPC1(DDE) in state 2
Map dataprimary map
Sample
  • Complex: AtTPC1(DDE)
    • Protein or peptide: Two pore calcium channel protein 1
  • Ligand: CALCIUM IONCalcium
  • Ligand: PALMITIC ACID
  • 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. / Ion transport domain / Ion transport protein / EF-hand domain / 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: 3.7 Å
AuthorsGreen EM / Kintzer AF / Stroud RM / Cheng Y
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM24485 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM098672 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
DepositionJul 10, 2018-
Header (metadata) releaseJul 25, 2018-
Map releaseSep 19, 2018-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 8
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6e1p
  • Surface level: 8
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8960.png

Downloads & links

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Map

FileDownload / File: emd_8960.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationprimary map
Voxel sizeX=Y=Z: 1.2156 Å
Density
Contour LevelBy AUTHOR: 8 / Movie #1: 8
Minimum - Maximum-26.79141 - 41.256287
Average (Standard dev.)-0.0000000096 (±0.9999997)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 311.1936 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21560156251.21560156251.2156015625
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z311.194311.194311.194
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-26.79141.256-0.000

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Supplemental data

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Half map: em-half-volume P1

Fileemd_8960_half_map_1.map
Annotationem-half-volume_P1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-volume P2

Fileemd_8960_half_map_2.map
Annotationhalf-volume_P2
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(DDE)

EntireName: AtTPC1(DDE)
Components
  • Complex: AtTPC1(DDE)
    • Protein or peptide: Two pore calcium channel protein 1
  • Ligand: CALCIUM IONCalcium
  • Ligand: PALMITIC ACID
  • Ligand: water

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Supramolecule #1: AtTPC1(DDE)

SupramoleculeName: AtTPC1(DDE) / 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

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: 84.547203 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MGGGGTDRVR R(SEP)EAI(TPO)HG(TPO)P FQKAAALVDL AEDGIGLPVE ILDQSSFGES ARYYFIFTRL DLIWSLNY F ALLFLNFFEQ PLWCEKNPKP SCKDRDYYYL GELPYLTNAE SIIYEVITLA ILLVHTFFPI SYEGSRIFWT SRLNLVKVA CVVILFVDVL ...String:
MGGGGTDRVR R(SEP)EAI(TPO)HG(TPO)P FQKAAALVDL AEDGIGLPVE ILDQSSFGES ARYYFIFTRL DLIWSLNY F ALLFLNFFEQ PLWCEKNPKP SCKDRDYYYL GELPYLTNAE SIIYEVITLA ILLVHTFFPI SYEGSRIFWT SRLNLVKVA CVVILFVDVL VDFLYLSPLA FDFLPFRIAP YVRVIIFILS IRELRDTLVL LSGMLGTYLN ILALWMLFLL FASWIAFVMF ENTQQGLTV FTSYGATLYQ MFILFTTSNN PDVWIPAYKS SRWSSVFFVL YVLIGVYFVT NLILAVVYDS FKEQLAKQVS G MDQMKRRM LEKAFGLIDS DKNGEIDKNQ CIKLFEQLTN YRTLPKISKE EFGLIFDELD DTRDFKINKD EFADLCQAIA LR FQKEEVP SLFEHFPQIY HSALSQQLRA FVRSPNFGYA ISFILIINFI AVVVETTLNI EESSAQKPWQ VAEFVFGWIY VLE MALKIY TYGFENYWRE GANRFDFLVT WVIVIGETAT FITPDENTFF SNGQWIRYLL LARMLRLIRL LMNVQRYRAF IATF ITLIP SLMPYLGTIF CVLCIYCSIG VQVFGGLVNA GNKKLFETEL AEDDYLLFNF NDYPNGMVTL FNLLVMGNWQ VWMES YKDL TGTWWSITYF VSFYVITILL LLNLVVAFVL EAFFTELDLE EEEKCQGQDS QEKRNRRRSA GSKSRSQRVD TLLHHM LGD ELSKPECSTS DTLVPR

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 5 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 3 / Number of copies: 16 / Formula: PLM
Molecular weightTheoretical: 256.424 Da
Chemical component information

ChemComp-PLM:
PALMITIC ACID / Palmitic acid

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 2 / 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

Concentration0.8 mg/mL
BufferpH: 7.3
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 20 K / Instrument: FEI VITROBOT MARK III
DetailsSample reconstituted into saposin A.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 41132 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 31000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 2-60 / Number grids imaged: 2 / Number real images: 3408 / Average exposure time: 0.2 sec. / Average electron dose: 1.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 996035
CTF correctionSoftware: (Name: Gctf, RELION)
Startup modelType of model: OTHER / Details: Ab Initio model in cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1.0) / Number images used: 67308
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6e1p:
Structure of AtTPC1(DDE) in state 2

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