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- EMDB-1761: Single particle analysis of PSD-95 in negative stain -

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Basic information

Entry
Database: EMDB / ID: EMD-1761
TitleSingle particle analysis of PSD-95 in negative stain
Map data3d map of PSD-95
Sample
  • Sample: Rat PSD-95
  • Protein or peptide: PSD-95
KeywordsScaffold protein / Membrane associated Guanylate Kinase / PDZ / SH3
Function / homology
Function and homology information


RHO GTPases activate CIT / positive regulation of AMPA glutamate receptor clustering / neuronal ion channel clustering / P2Y1 nucleotide receptor binding / Neurexins and neuroligins / beta-1 adrenergic receptor binding / neuroligin family protein binding / structural constituent of postsynaptic density / synaptic vesicle maturation / positive regulation of neuron projection arborization ...RHO GTPases activate CIT / positive regulation of AMPA glutamate receptor clustering / neuronal ion channel clustering / P2Y1 nucleotide receptor binding / Neurexins and neuroligins / beta-1 adrenergic receptor binding / neuroligin family protein binding / structural constituent of postsynaptic density / synaptic vesicle maturation / positive regulation of neuron projection arborization / regulation of grooming behavior / receptor localization to synapse / protein localization to synapse / vocalization behavior / cerebellar mossy fiber / proximal dendrite / LGI-ADAM interactions / cellular response to potassium ion / Trafficking of AMPA receptors / dendritic branch / neuron spine / negative regulation of receptor internalization / Activation of Ca-permeable Kainate Receptor / AMPA glutamate receptor clustering / dendritic spine morphogenesis / establishment or maintenance of epithelial cell apical/basal polarity / frizzled binding / juxtaparanode region of axon / dendritic spine organization / neuron projection terminus / postsynaptic neurotransmitter receptor diffusion trapping / acetylcholine receptor binding / Synaptic adhesion-like molecules / positive regulation of synapse assembly / regulation of NMDA receptor activity / RAF/MAP kinase cascade / positive regulation of dendrite morphogenesis / beta-2 adrenergic receptor binding / neurotransmitter receptor localization to postsynaptic specialization membrane / cortical cytoskeleton / extrinsic component of cytoplasmic side of plasma membrane / locomotory exploration behavior / regulation of neuronal synaptic plasticity / kinesin binding / AMPA glutamate receptor complex / social behavior / excitatory synapse / neuromuscular process controlling balance / positive regulation of protein tyrosine kinase activity / positive regulation of excitatory postsynaptic potential / D1 dopamine receptor binding / Unblocking of NMDA receptors, glutamate binding and activation / glutamate receptor binding / positive regulation of synaptic transmission / ionotropic glutamate receptor binding / dendrite cytoplasm / cell periphery / synaptic membrane / PDZ domain binding / postsynaptic density membrane / regulation of long-term neuronal synaptic plasticity / establishment of protein localization / neuromuscular junction / kinase binding / cerebral cortex development / cell-cell adhesion / cell-cell junction / cell junction / synaptic vesicle / positive regulation of cytosolic calcium ion concentration / scaffold protein binding / protein-containing complex assembly / basolateral plasma membrane / chemical synaptic transmission / protein phosphatase binding / postsynaptic membrane / postsynapse / dendritic spine / postsynaptic density / neuron projection / signaling receptor binding / glutamatergic synapse / dendrite / synapse / protein-containing complex binding / protein kinase binding / endoplasmic reticulum / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Polyubiquitination (PEST) N-terminal domain of MAGUK / Disks large homologue 1, N-terminal PEST domain / Polyubiquitination (PEST) N-terminal domain of MAGUK / PDZ-associated domain of NMDA receptors / PDZ-associated domain of NMDA receptors / Disks large 1-like / Disks large 1-like / : / Guanylate kinase, conserved site / Guanylate kinase-like signature. ...Polyubiquitination (PEST) N-terminal domain of MAGUK / Disks large homologue 1, N-terminal PEST domain / Polyubiquitination (PEST) N-terminal domain of MAGUK / PDZ-associated domain of NMDA receptors / PDZ-associated domain of NMDA receptors / Disks large 1-like / Disks large 1-like / : / Guanylate kinase, conserved site / Guanylate kinase-like signature. / Guanylate kinase-like domain profile. / Guanylate kinase-like domain / Guanylate kinase/L-type calcium channel beta subunit / Guanylate kinase / Guanylate kinase homologues. / PDZ domain / SH3 domain / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Src homology 3 domains / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Disks large homolog 4
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / negative staining / Resolution: 22.9 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 13, 2010-
Header (metadata) releaseMar 11, 2011-
Map releaseJul 14, 2011-
UpdateSep 19, 2012-
Current statusSep 19, 2012Processing 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 radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2xkx
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_1761.png

Downloads & links

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Map

FileDownload / File: emd_1761.map.gz / Format: CCP4 / Size: 422.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3d map of PSD-95
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.67 Å/pix.
x 48 pix.
= 176.016 Å		3.67 Å/pix.
x 48 pix.
= 176.016 Å		3.67 Å/pix.
x 48 pix.
= 176.016 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.667 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-1.88844132 - 3.87746572
Average (Standard dev.)0.0 (±0.303009)
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-1.8883.8770.000

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Supplemental data

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Sample components

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Entire : Rat PSD-95

EntireName: Rat PSD-95
Components
  • Sample: Rat PSD-95
  • Protein or peptide: PSD-95

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Supramolecule #1000: Rat PSD-95

SupramoleculeName: Rat PSD-95 / type: sample / ID: 1000 / Oligomeric state: Monomer / Number unique components: 1
Molecular weightExperimental: 95 KDa / Theoretical: 78 KDa / Method: SDS-PAGE

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Macromolecule #1: PSD-95

MacromoleculeName: PSD-95 / type: protein_or_peptide / ID: 1 / Name.synonym: PSD-95 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Norway Rat / Location in cell: Membrane
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, 5mM DTT, 1mM EDTA,
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: GENERIC FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 15 µm / Number real images: 2 / Details: none / Od range: 2 / 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: 3.6 mm / Nominal defocus max: 2.25 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 43000
Sample stageSpecimen holder: Eucentric / Specimen holder model: PHILIPS ROTATION HOLDER

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Image processing

DetailsParticles selected using model based picking using an ab-initio model from Small angle X-ray scattering
CTF correctionDetails: Paramters determined using Scattering curve
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 22.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 7854
Final two d classificationNumber classes: 84

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Atomic model buiding 1

Initial modelPDB ID:

1iu0

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

PDB-2xkx:
Single particle analysis of PSD-95 in negative stain

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Atomic model buiding 2

Initial modelPDB ID:

1qlc

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

PDB-2xkx:
Single particle analysis of PSD-95 in negative stain

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Atomic model buiding 3

Initial modelPDB ID:

1tp5

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

PDB-2xkx:
Single particle analysis of PSD-95 in negative stain

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Atomic model buiding 4

Initial modelPDB ID:

1kjw

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

PDB-2xkx:
Single particle analysis of PSD-95 in negative stain

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