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- PDB-6v1q: Two-pore channel 3 -

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Basic information

Entry
Database: PDB / ID: 6v1q
TitleTwo-pore channel 3
ComponentsTwo pore channel 3
KeywordsMEMBRANE PROTEIN / Voltag-gated ion channel / two-pore channel / TPC3
Function / homologyIon transport domain / Voltage-dependent channel domain superfamily / Ion transport protein / ion channel activity / integral component of membrane / Two pore channel 3
Function and homology information
Biological speciesDanio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsDickinson, M.S. / Stroud, R.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM24485 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Resting state structure of the hyperdepolarization activated two-pore channel 3.
Authors: Miles Sasha Dickinson / Alexander Myasnikov / Jacob Eriksen / Nicole Poweleit / Robert M Stroud /
Abstract: Voltage-gated ion channels endow membranes with excitability and the means to propagate action potentials that form the basis of all neuronal signaling. We determined the structure of a voltage-gated ...Voltage-gated ion channels endow membranes with excitability and the means to propagate action potentials that form the basis of all neuronal signaling. We determined the structure of a voltage-gated sodium channel, two-pore channel 3 (TPC3), which generates ultralong action potentials. TPC3 is distinguished by activation only at extreme membrane depolarization (V ∼ +75 mV), in contrast to other TPCs and Na channels that activate between -20 and 0 mV. We present electrophysiological evidence that TPC3 voltage activation depends only on voltage sensing domain 2 (VSD2) and that each of the three gating arginines in VSD2 reduces the activation threshold. The structure presents a chemical basis for sodium selectivity, and a constricted gate suggests a closed pore consistent with extreme voltage dependence. The structure, confirmed by our electrophysiology, illustrates the configuration of a bona fide resting state voltage sensor, observed without the need for any inhibitory ligand, and independent of any chemical or mutagenic alteration.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 20, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 4, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 8, 2020Group: Author supporting evidence / Database references / Category: citation / pdbx_audit_support
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year / _pdbx_audit_support.funding_organization
Revision 1.2Jan 22, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Feb 12, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Two pore channel 3
B: Two pore channel 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)177,5713
Polymers177,5482
Non-polymers231
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Two pore channel 3 / Two pore segment channel 3


Mass: 88774.164 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: tpcn3, gb:eh507706, TPC3 / Production host: Homo sapiens (human) / References: UniProt: C4IXV6
#2: Chemical ChemComp-NA / SODIUM ION / Sodium


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Dimer of two-pore channel 3 subunits / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Danio rerio (zebrafish)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
Buffer component
IDConc.FormulaBuffer-ID
120 mMTris1
2150 mMNaClSodium chloride1
30.06 %GDN1
SpecimenConc.: 3 mg/ml / 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 IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 92 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
12cryoSPARC23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 213328 / Symmetry type: POINT

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