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- PDB-6e1k: Structure of AtTPC1(DDE) reconstituted in saposin A with cat06 Fab -

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Basic information

Entry
Database: PDB / ID: 6e1k
TitleStructure of AtTPC1(DDE) reconstituted in saposin A with cat06 Fab
Components
  • Two pore calcium channel protein 1
  • cat06 heavy chain
  • cat06 light chain
KeywordsMEMBRANE PROTEIN / Two-pore channel
Function / homology
Function and homology information


regulation of jasmonic acid biosynthetic process / plant-type vacuole / seed germination / regulation of stomatal movement / vacuolar membrane / vacuole / voltage-gated calcium channel activity / regulation of ion transmembrane transport / calcium ion transport / calcium-mediated signaling ...regulation of jasmonic acid biosynthetic process / plant-type vacuole / seed germination / regulation of stomatal movement / vacuolar membrane / vacuole / voltage-gated calcium channel activity / regulation of ion transmembrane transport / calcium ion transport / calcium-mediated signaling / calcium ion binding / Golgi apparatus / integral component of membrane / identical protein binding / plasma membrane / cytosol
Ion transport protein / Voltage-dependent channel domain superfamily / EF-hand domain pair / Ion transport domain / EF-hand domain
Two pore calcium channel protein 1
Biological speciesArabidopsis thaliana (thale cress)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKintzer, A.F. / Green, E.M. / Cheng, Y. / Stroud, R.M.
Funding support United States, 2items
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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJul 10, 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 1.2Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: Two pore calcium channel protein 1
B: Two pore calcium channel protein 1
C: cat06 light chain
D: cat06 heavy chain
E: cat06 light chain
F: cat06 heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)268,88227
Polymers265,0116
Non-polymers3,87021
Water362
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 3 types, 6 molecules ABCEDF

#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: 84547.203 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: TPC1, CCH1, FOU2, At4g03560, F9H3.19, T5L23.5 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q94KI8
#2: Protein cat06 light chain


Mass: 23171.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Protein cat06 heavy chain


Mass: 24786.658 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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Non-polymers , 3 types, 23 molecules

#4: Chemical
ChemComp-CA / CALCIUM ION / Calcium


Mass: 40.078 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Ca
#5: Chemical
ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C16H32O2
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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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(DDE) reconstituted in saposin A / Type: COMPLEX / Entity ID: 1,2,3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Buffer solutionpH: 7.3
SpecimenConc.: 0.8 mg/ml / Details: Sample reconstituted into saposin A. / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 20 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 31000 X / Calibrated magnification: 41132 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 1 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3408
Image scansMovie frames/image: 60 / Used frames/image: 2-60

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4GctfCTF correction
5RELIONCTF correction
10PHENIX1.13-2998model refinement
11RELION2.1.0initial Euler assignment
12RELION2.1.0final Euler assignment
14RELION2.1.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 996035
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 224577 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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