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- EMDB-1765: Single particle analysis of the 1:1 complex of PSD-95 and Kir2.1N... -

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Basic information

Entry
Database: EMDB / ID: EMD-1765
TitleSingle particle analysis of the 1:1 complex of PSD-95 and Kir2.1NC_4 in negative stain
Map dataNegative stain EM map of the complex of PSD-95 and Kir2.1NC
Sample
  • Sample: Complex of mouse Kir2.1, cytoplamic domain, homotetramer of fused N,C termini with rat PSD-95
  • Protein or peptide: Kir2.1 cytoplasmic domain
  • Protein or peptide: PSD-95DLG4
KeywordsScaffold protein / membrane associated / Guanylatekinase / PDZ / SH3 / Ion Channel / cytoplasmic domain / inwardly rectifying / membrane protein / homotetramer
Function / homologyPotassium channel, inwardly rectifying, Kir2.1 / neuronal ion channel clustering / Disks large 1-like / membrane => GO:0016020
Function and homology information
Biological speciesMus musculus (house mouse) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / negative staining / Resolution: 20.5 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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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image1765.png

Downloads & links

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Map

FileDownload / File: emd_1765.map.gz / Format: CCP4 / Size: 422.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain EM map of the complex of PSD-95 and Kir2.1NC
Voxel sizeX=Y=Z: 3.667 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-0.7391082 - 5.22309399
Average (Standard dev.)0.0 (±0.42598441)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-24-24-24
Dimensions484848
Spacing484848
CellA=B=C: 176.016 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.6673.6673.667
M x/y/z484848
origin x/y/z0.0000.0000.000
length x/y/z176.016176.016176.016
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ454586
MAP C/R/S123
start NC/NR/NS-24-24-24
NC/NR/NS484848
D min/max/mean-0.7395.223-0.000

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Supplemental data

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Sample components

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Entire : Complex of mouse Kir2.1, cytoplamic domain, homotetramer of fused...

EntireName: Complex of mouse Kir2.1, cytoplamic domain, homotetramer of fused N,C termini with rat PSD-95
Components
  • Sample: Complex of mouse Kir2.1, cytoplamic domain, homotetramer of fused N,C termini with rat PSD-95
  • Protein or peptide: Kir2.1 cytoplasmic domain
  • Protein or peptide: PSD-95DLG4

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Supramolecule #1000: Complex of mouse Kir2.1, cytoplamic domain, homotetramer of fused...

SupramoleculeName: Complex of mouse Kir2.1, cytoplamic domain, homotetramer of fused N,C termini with rat PSD-95
type: sample / ID: 1000
Details: Multi-refinement was undertaken to separate Kir2.1NC_4 alone from the complex
Oligomeric state: One tetramer of Kir2.1NC binds to PSD-95 / Number unique components: 2
Molecular weightTheoretical: 218 KDa / Method: Sum of component MW

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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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Macromolecule #2: PSD-95

MacromoleculeName: PSD-95 / type: protein_or_peptide / ID: 2 / Name.synonym: PSD-95 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Rat
Molecular weightExperimental: 95 KDa / Theoretical: 78 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pGEX-6P
SequenceGO: neuronal ion channel clustering / InterPro: Disks large 1-like

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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
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 3.6 mm / Nominal defocus max: 1.75 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 43000
Sample stageSpecimen holder: Eucentric / Specimen holder model: PHILIPS ROTATION HOLDER
DetailsLow dose
Image recordingCategory: FILM / Film or detector model: GENERIC FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 15 µm / Number real images: 15 / Od range: 2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: Parameters determined using a calculated Scattering curve
Final two d classificationNumber classes: 84
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 20.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 24199
DetailsParticles initially selected using automated particle picking based on a subset of representative particles on each micrograph.

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Atomic model buiding 1

Initial modelPDB ID:

2xky

SoftwareName: Chimera
DetailsROTATION MATRIX 0.62166 0.76947 -0.14647 0.00842 0.18041 0.98355 0.78325 -0.61267 0.10567 TRANSLATION VECTOR IN AS -7.89876 -3.87420 -3.22822
RefinementSpace: REAL

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Atomic model buiding 2

Initial modelPDB ID:

2xkx

SoftwareName: Chimera
DetailsROTATION MATRIX 0.31682 0.16747 0.93358 -0.39203 -0.87316 0.28967 0.86368 -0.45776 -0.21099 TRANSLATION VECTOR IN AS -6.39213 21.17728 3.96066
RefinementSpace: REAL

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