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- EMDB-3063: Cryo-EM structure of the Slo2.2 Na+-activated K+ channel -

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Basic information

Entry
Database: EMDB / ID: EMD-3063
TitleCryo-EM structure of the Slo2.2 Na+-activated K+ channel
Map dataFocus refined reconstruction of chicken Slo2.2 gating ring
Sample
  • Sample: chicken Slo2.2
  • Protein or peptide: Slo2.2
KeywordsIon channel / potassium channel
Function / homology
Function and homology information


intracellular sodium-activated potassium channel activity / outward rectifier potassium channel activity / potassium ion transmembrane transport / plasma membrane
Similarity search - Function
Calcium-activated potassium channel BK, alpha subunit / : / Calcium-activated BK potassium channel alpha subunit / Calcium-activated potassium channel slowpoke-like RCK domain / RCK N-terminal domain profile. / Potassium channel domain / Ion channel
Similarity search - Domain/homology
Potassium channel subfamily T member 1
Similarity search - Component
Biological speciesGallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsHite RK / Yuan P / Li Z / Hsuing Y / Walz T / MacKinnon R
CitationJournal: Nature / Year: 2015
Title: Cryo-electron microscopy structure of the Slo2.2 Na(+)-activated K(+) channel.
Authors: Richard K Hite / Peng Yuan / Zongli Li / Yichun Hsuing / Thomas Walz / Roderick MacKinnon /
Abstract: Na(+)-activated K(+) channels are members of the Slo family of large conductance K(+) channels that are widely expressed in the brain, where their opening regulates neuronal excitability. These ...Na(+)-activated K(+) channels are members of the Slo family of large conductance K(+) channels that are widely expressed in the brain, where their opening regulates neuronal excitability. These channels fulfil a number of biological roles and have intriguing biophysical properties, including conductance levels that are ten times those of most other K(+) channels and gating sensitivity to intracellular Na(+). Here we present the structure of a complete Na(+)-activated K(+) channel, chicken Slo2.2, in the Na(+)-free state, determined by cryo-electron microscopy at a nominal resolution of 4.5 ångströms. The channel is composed of a large cytoplasmic gating ring, in which resides the Na(+)-binding site and a transmembrane domain that closely resembles voltage-gated K(+) channels. In the structure, the cytoplasmic domain adopts a closed conformation and the ion conduction pore is also closed. The structure reveals features that can explain the unusually high conductance of Slo channels and how contraction of the cytoplasmic gating ring closes the pore.
History
DepositionJun 25, 2015-
Header (metadata) releaseAug 12, 2015-
Map releaseOct 14, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5a6f
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5a6f
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3063.png

Downloads & links

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Map

FileDownload / File: emd_3063.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocus refined reconstruction of chicken Slo2.2 gating ring
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 256 pix.
= 266.24 Å		1.04 Å/pix.
x 256 pix.
= 266.24 Å		1.04 Å/pix.
x 256 pix.
= 266.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.01296597 - 0.03799237
Average (Standard dev.)-0.000052 (±0.00295178)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 266.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z266.240266.240266.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0130.038-0.000

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Supplemental data

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Sample components

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Entire : chicken Slo2.2

EntireName: chicken Slo2.2
Components
  • Sample: chicken Slo2.2
  • Protein or peptide: Slo2.2

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Supramolecule #1000: chicken Slo2.2

SupramoleculeName: chicken Slo2.2 / type: sample / ID: 1000 / Oligomeric state: tetramer / Number unique components: 1
Molecular weightTheoretical: 550 KDa

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Macromolecule #1: Slo2.2

MacromoleculeName: Slo2.2 / type: protein_or_peptide / ID: 1 / Name.synonym: Slack, KCNT1 / Number of copies: 4 / Oligomeric state: tetramer / Recombinant expression: Yes
Source (natural)Organism: Gallus gallus (chicken) / synonym: chicken / Location in cell: Plasma membrane
Molecular weightExperimental: 550 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9 / Recombinant plasmid: pFastbac
SequenceUniProtKB: Potassium channel subfamily T member 1

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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

Concentration4.0 mg/mL
BufferpH: 7.4
Details: 20 mM HEPES pH 7.4, 300 mM KCl, 1.5 mM dodecyl maltoside, 0.05 mg/ml POPE:POPG (3:1)
GridDetails: 300 mesh Cu Quantifoil R1.2 / 1.3
VitrificationCryogen name: ETHANE / Chamber humidity: 84 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV / Method: 4 second blot prior to plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Alignment procedureLegacy - Astigmatism: FEI Image corrector
Specialist opticsEnergy filter - Name: Gatan / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 30.0 eV
DateJul 7, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 2243 / Average electron dose: 40 e/Å2
Details: 25 sub-frames were recorded for each image in super-resolution counting mode.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsManually picked particles with BOXER. 2D and 3D classification in RELION. Final map generated with RELION using a soft mask that includes only the gating ring.
CTF correctionDetails: Each Image
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: OTHER / Software - Name: SPARX, RELION / Number images used: 24231
FSC plot (resolution estimation)

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