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- EMDB-21015: Two-pore channel 3 -

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Basic information

Entry
Database: EMDB / ID: EMD-21015
TitleTwo-pore channel 3
Map dataUnsharpened map from non-uniform refinement in cryoSPARC v2.
Sample
  • Complex: Dimer of two-pore channel 3 subunits
    • Protein or peptide: Two pore channel 3
  • Ligand: SODIUM IONSodium
KeywordsVoltag-gated ion channel / two-pore channel / TPC3 / MEMBRANE PROTEIN
Function / homology
Function and homology information


sodium ion transmembrane transporter activity / voltage-gated sodium channel activity / action potential / plasma membrane => GO:0005886 / sodium ion transmembrane transport / protein homodimerization activity
Similarity search - Function
Voltage-dependent channel domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Biological speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.11 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)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.
History
DepositionNov 20, 2019-
Header (metadata) releaseDec 4, 2019-
Map releaseDec 4, 2019-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.863
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.863
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6v1q
  • Surface level: 0.863
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21015.png

Downloads & links

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Map

FileDownload / File: emd_21015.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map from non-uniform refinement in cryoSPARC v2.
Voxel sizeX=Y=Z: 1.256 Å
Density
Contour LevelBy AUTHOR: 0.863 / Movie #1: 0.863
Minimum - Maximum-0.64165455 - 2.344392
Average (Standard dev.)-0.0008597384 (±0.0817106)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 351.68002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2561.2561.256
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z351.680351.680351.680
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.6422.344-0.001

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Supplemental data

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Additional map: Locally sharpened map from non-uniform refinement in cryoSPARC...

Fileemd_21015_additional.map
AnnotationLocally sharpened map from non-uniform refinement in cryoSPARC v2, used for atomic modeling.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimer of two-pore channel 3 subunits

EntireName: Dimer of two-pore channel 3 subunits
Components
  • Complex: Dimer of two-pore channel 3 subunits
    • Protein or peptide: Two pore channel 3
  • Ligand: SODIUM IONSodium

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Supramolecule #1: Dimer of two-pore channel 3 subunits

SupramoleculeName: Dimer of two-pore channel 3 subunits / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Danio rerio (zebrafish)

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Macromolecule #1: Two pore channel 3

MacromoleculeName: Two pore channel 3 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 88.774164 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSEGKTEKTS HTLTKDEGFT NGGNHVPSNV TDQMTEKFDL ATVYVSDAKY NRNIFFDTSP QAVKLYLLYN HWFMQTLVYV FIIINLALA LFEDPAVVPL PIWATSTIET ICLSAFTVRI IHYAKVIPKD KFWKDPKNIC IIIIVTLSFI DMVIYGALKA T GHYGIRWS ...String:
MSEGKTEKTS HTLTKDEGFT NGGNHVPSNV TDQMTEKFDL ATVYVSDAKY NRNIFFDTSP QAVKLYLLYN HWFMQTLVYV FIIINLALA LFEDPAVVPL PIWATSTIET ICLSAFTVRI IHYAKVIPKD KFWKDPKNIC IIIIVTLSFI DMVIYGALKA T GHYGIRWS RVLRPLLLVN VTEGRQLRRA FRSIRNALPQ ISYVFFLFMF SVLVFSLMAL KLFGKRGLLT INGSPYFTDY MD IVFDLYV LVTTANSPDV MMPAYNSSVY FTIFFILYIV INTYTFMSFF LAVVYNNYKK YLKEEVRQLV KAKRIKMCRA FSL LQENRG EGGEPVVTQA NWNHLVKLVK PKISTAHREL LWSVLDDQNK GHIGKFAFVQ LADLLSIQVI TVKSQAHPIQ ICFP SLYNS LPSRFIRQMV HHRVFVYAYD LIILVNAVFI GLDEENPVVS NAEWGFLALY MLEILLKLYA TEPRAFFARH QFWNW FDTI IVVSALFGTI INSALKHSGG YTSRQVLDIV FILRVLRLIR VVDSIKRFRA IINTLIKIGP TILTFGQLIL VVYYIF AMV GMELFKGKIQ FFEPNSTSPD REYCGNPLLK STSFAKLNYC KNNFNDVISS FILLLELTVV NQWHVLTSGF TAVTHVS AR LFFVIFHIVV VIIIINIFVA FILEAFLVEY TVDKSELQTS LEKKIEELEL NVQQDGVDTG LVDAMETNDS DLGSSEDG K RKPSLMFKIA SRRSRTVDGL LQRMFETDLR PEDFNEEELD NTNFSNPVFD SV

UniProtKB: Two pore channel 3

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Macromolecule #2: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 2 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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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

Concentration3 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormula
20.0 mMTris
150.0 mMNaClSodium chloride
0.06 %GDN
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 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: 70.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 92.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 213328

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