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- EMDB-1764: Single particle analysis of Kir2.1NC_4 in negative stain -

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Basic information

Entry
Database: EMDB / ID: EMD-1764
TitleSingle particle analysis of Kir2.1NC_4 in negative stain
Map data3d map of the Kir2.1NC_4 tetramer, C_4 symmetry applied
Sample
  • Sample: mouse Kir2.1, cytoplamic domain, homotetramer of fused N,C termini
  • Protein or peptide: Kir2.1 cytoplasmic domain
KeywordsIon Channel / cytoplasmic domain / inwardly rectifying / membrane protein / homotetramer
Function / homology
Function and homology information


Classical Kir channels / regulation of skeletal muscle contraction via regulation of action potential / relaxation of skeletal muscle / Phase 4 - resting membrane potential / cardiac muscle cell action potential / magnesium ion transport / voltage-gated potassium channel activity involved in cardiac muscle cell action potential repolarization / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / regulation of membrane repolarization ...Classical Kir channels / regulation of skeletal muscle contraction via regulation of action potential / relaxation of skeletal muscle / Phase 4 - resting membrane potential / cardiac muscle cell action potential / magnesium ion transport / voltage-gated potassium channel activity involved in cardiac muscle cell action potential repolarization / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / regulation of membrane repolarization / inward rectifier potassium channel activity / positive regulation of potassium ion transmembrane transport / cardiac muscle cell action potential involved in contraction / regulation of cardiac muscle cell contraction / relaxation of cardiac muscle / potassium ion import across plasma membrane / regulation of heart rate by cardiac conduction / intercalated disc / membrane => GO:0016020 / phosphatidylinositol-4,5-bisphosphate binding / voltage-gated potassium channel complex / T-tubule / cellular response to mechanical stimulus / potassium ion transport / protein homotetramerization / dendritic spine / postsynaptic membrane / neuronal cell body / glutamatergic synapse / identical protein binding / membrane / plasma membrane
Similarity search - Function
Potassium channel, inwardly rectifying, Kir2.1 / Potassium channel, inwardly rectifying, Kir2.1 / Potassium channel, inwardly rectifying, Kir, N-terminal / Inward rectifier potassium channel N-terminal / Potassium channel, inwardly rectifying, transmembrane domain / Inward rectifier potassium channel transmembrane domain / Potassium channel, inwardly rectifying, Kir, cytoplasmic / Potassium channel, inwardly rectifying, Kir / Inward rectifier potassium channel, C-terminal / Inward rectifier potassium channel C-terminal domain / Immunoglobulin E-set
Similarity search - Domain/homology
Inward rectifier potassium channel 2
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 17.2 Å
AuthorsFomina S / Howard TD / Sleator OK / Golovanova M / O'Ryan L / Leyland M / Grossmann JG / Collins RF / Prince SM
CitationJournal: Biochim Biophys Acta / Year: 2011
Title: Self-directed assembly and clustering of the cytoplasmic domains of inwardly rectifying Kir2.1 potassium channels on association with PSD-95.
Authors: Svetlana Fomina / Tina D Howard / Olivia K Sleator / Marina Golovanova / Liam O'Ryan / Mark L Leyland / J Günter Grossmann / Richard F Collins / Stephen M Prince /
Abstract: The interaction of the extra-membranous domain of tetrameric inwardly rectifying Kir2.1 ion channels (Kir2.1NC(4)) with the membrane associated guanylate kinase protein PSD-95 has been studied using ...The interaction of the extra-membranous domain of tetrameric inwardly rectifying Kir2.1 ion channels (Kir2.1NC(4)) with the membrane associated guanylate kinase protein PSD-95 has been studied using Transmission Electron Microscopy in negative stain. Three types of complexes were observed in electron micrographs corresponding to a 1:1 complex, a large self-enclosed tetrad complex and extended chains of linked channel domains. Using models derived from small angle X-ray scattering experiments in which high resolution structures from X-ray crystallographic and Nuclear Magnetic Resonance studies are positioned, the envelopes from single particle analysis can be resolved as a Kir2.1NC(4):PSD-95 complex and a tetrad of this unit (Kir2.1NC(4):PSD-95)(4). The tetrad complex shows the close association of the Kir2.1 cytoplasmic domains and the influence of PSD-95 mediated self-assembly on the clustering of these channels.
History
DepositionJul 15, 2010-
Header (metadata) releaseMar 11, 2011-
Map releaseJul 14, 2011-
UpdateNov 26, 2014-
Current statusNov 26, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2xky
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
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Supplemental images

emd_1764.png

Downloads & links

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Map

FileDownload / File: emd_1764.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3d map of the Kir2.1NC_4 tetramer, C_4 symmetry applied
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.93 Å/pix.
x 64 pix.
= 187.52 Å		2.93 Å/pix.
x 64 pix.
= 187.52 Å		2.93 Å/pix.
x 64 pix.
= 187.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-1.18297374 - 3.58607507
Average (Standard dev.)0.0 (±0.32558325)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 187.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.932.932.93
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z187.520187.520187.520
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ454586
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-1.1833.5860.000

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Supplemental data

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Sample components

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Entire : mouse Kir2.1, cytoplamic domain, homotetramer of fused N,C termini

EntireName: mouse Kir2.1, cytoplamic domain, homotetramer of fused N,C termini
Components
  • Sample: mouse Kir2.1, cytoplamic domain, homotetramer of fused N,C termini
  • Protein or peptide: Kir2.1 cytoplasmic domain

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Supramolecule #1000: mouse Kir2.1, cytoplamic domain, homotetramer of fused N,C termini

SupramoleculeName: mouse Kir2.1, cytoplamic domain, homotetramer of fused N,C termini
type: sample / ID: 1000 / Oligomeric state: Tetramer / Number unique components: 1
Molecular weightExperimental: 140 KDa / Theoretical: 140 KDa / Method: SDS-PAGE for Kir2.1NC construct x 4

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Macromolecule #1: Kir2.1 cytoplasmic domain

MacromoleculeName: Kir2.1 cytoplasmic domain / type: protein_or_peptide / ID: 1 / Name.synonym: Kir2.1NC / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: Mouse / Location in cell: Membrane
Molecular weightExperimental: 140 KDa / Theoretical: 140 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET15b
SequenceGO: membrane => GO:0016020 / InterPro: Potassium channel, inwardly rectifying, Kir2.1

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Details: 20mM Tris/HCl, 150mM NaCl, 1mM reduced GSH, 1mM EDTA, 50mM L-Glutamic acid, 50mM L-Arginine
StainingType: NEGATIVE
Details: Samples were adsorbed onto freshly glow discharged carbon film grids. Sample solution was pipetted into the grid followed by blotting, de-ionized water was then applied for 10s followed by ...Details: Samples were adsorbed onto freshly glow discharged carbon film grids. Sample solution was pipetted into the grid followed by blotting, de-ionized water was then applied for 10s followed by blotting, 2% w/v Uranyl Acetate solution was applied followed by a final blotting step.
GridDetails: 400 mesh Copper
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 10
DetailsLow dose
Image recordingCategory: FILM / Film or detector model: GATAN ORIUS SC200 (2k x 2k) / Digitization - Scanner: OTHER / Digitization - Sampling interval: 10 µm / Number real images: 22 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 1.25 µm / Nominal defocus min: 0.6 µm
Sample stageSpecimen holder: Eucentric / Specimen holder model: JEOL

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Image processing

DetailsParticles initially selected using automated particle picking based on a subset of representative particles on a single micrograph, followed by model-based picking.
CTF correctionDetails: Parameters determined using Scattering curve
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 17.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 49012
Final two d classificationNumber classes: 62

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Atomic model buiding 1

Initial modelPDB ID:

1u4f

SoftwareName: Chimera
DetailsTetramer model assembled using SAXS refinement and docked
RefinementSpace: REAL
Output model

PDB-2xky:
Single particle analysis of Kir2.1NC_4 in negative stain

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