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

Basic information

• Entry: Database: PDB / ID: 2xkx
• Title: Single particle analysis of PSD-95 in negative stain
• Components: DISKS LARGE HOMOLOG 4
• Keywords: STRUCTURAL PROTEIN / SCAFFOLD PROTEIN / MEMBRANE ASSOCIATED GUANYLATE KINASE

Function / homology

Function:

RHO GTPases activate CIT / positive regulation of AMPA glutamate receptor clustering / neuronal ion channel clustering / P2Y1 nucleotide receptor binding / beta-1 adrenergic receptor binding / Neurexins and neuroligins / neuroligin family protein binding / receptor localization to synapse / positive regulation of neuron projection arborization / regulation of grooming behavior / structural constituent of postsynaptic density / synaptic vesicle maturation / proximal dendrite / AMPA glutamate receptor clustering / cellular response to potassium ion / cerebellar mossy fiber / protein localization to synapse / vocalization behavior / LGI-ADAM interactions / neuron spine / Trafficking of AMPA receptors / dendritic branch / Activation of Ca-permeable Kainate Receptor / juxtaparanode region of axon / neuron projection terminus / establishment or maintenance of epithelial cell apical/basal polarity / dendritic spine morphogenesis / negative regulation of receptor internalization / postsynaptic neurotransmitter receptor diffusion trapping / frizzled binding / dendritic spine organization / acetylcholine receptor binding / positive regulation of synapse assembly / RAF/MAP kinase cascade / Synaptic adhesion-like molecules / neurotransmitter receptor localization to postsynaptic specialization membrane / positive regulation of dendrite morphogenesis / beta-2 adrenergic receptor binding / regulation of neuronal synaptic plasticity / locomotory exploration behavior / cortical cytoskeleton / regulation of NMDA receptor activity / social behavior / positive regulation of excitatory postsynaptic potential / AMPA glutamate receptor complex / kinesin binding / neuromuscular process controlling balance / excitatory synapse / D1 dopamine receptor binding / Unblocking of NMDA receptors, glutamate binding and activation / glutamate receptor binding / positive regulation of protein tyrosine kinase activity / positive regulation of synaptic transmission / ionotropic glutamate receptor binding / extrinsic component of cytoplasmic side of plasma membrane / dendrite cytoplasm / synaptic membrane / PDZ domain binding / cell periphery / postsynaptic density membrane / regulation of long-term neuronal synaptic plasticity / neuromuscular junction / establishment of protein localization / cell-cell adhesion / cerebral cortex development / kinase binding / cell-cell junction / synaptic vesicle / cell junction / positive regulation of cytosolic calcium ion concentration / chemical synaptic transmission / postsynaptic membrane / scaffold protein binding / postsynapse / basolateral plasma membrane / protein-containing complex assembly / protein phosphatase binding / dendritic spine / postsynaptic density / neuron projection / signaling receptor binding / dendrite / glutamatergic synapse / synapse / protein-containing complex binding / protein kinase binding / endoplasmic reticulum / membrane / plasma membrane / cytosol / cytoplasm

Domain/homology:

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 / 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

Component:

Disks large homolog 4

• Biological species: RATTUS NORVEGICUS (Norway rat)
• Method: ELECTRON MICROSCOPY / SOLUTION SCATTERING / single particle reconstruction / negative staining / Resolution: 22.9 Å
• Model type details: CA ATOMS ONLY, CHAIN A, B
• Authors: Fomina, S. / Howard, T.D. / Sleator, O.K. / Golovanova, M. / O'Ryan, L. / Leyland, M.L. / Grossmann, J.G. / Collins, R.F. / Prince, S.M.

Citation

Journal: 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 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.

#1: Journal: J Mol Biol / Year: 2003
Title: Supramodular structure and synergistic target binding of the N-terminal tandem PDZ domains of PSD-95.
Authors: Jia-Fu Long / Hidehito Tochio / Ping Wang / Jing-Song Fan / Carlo Sala / Martin Niethammer / Morgan Sheng / Mingjie Zhang /
Abstract: PDZ domain proteins play critical roles in binding, clustering and subcellular targeting of membrane receptors and ion channels. PDZ domains in multi-PDZ proteins often are arranged in groups with highly conserved spacing and intervening sequences; however, the functional significance of such tandem arrangements of PDZs is unclear. We have solved the three-dimensional structure of the first two PDZ domains of postsynaptic density protein-95 (PSD-95 PDZ1 and PDZ2), which are closely linked to each other in the PSD-95 family of scaffold proteins. The two PDZs have limited freedom of rotation and their C-terminal peptide-binding grooves are aligned with each other with an orientation preference for binding to pairs of C termini extending in the same direction. Increasing the spacing between PDZ1 and PDZ2 resulted in decreased binding between PDZ12 and its dimeric targets. The same mutation impaired the functional ability of PSD-95 to cluster Kv1.4 potassium channels in heterologous cells. The data presented provide a molecular basis for preferential binding of PSD-95 to multimeric membrane proteins with appropriate C-terminal sequences.

#2: Journal: J Mol Biol / Year: 2000
Title: Solution structure and backbone dynamics of the second PDZ domain of postsynaptic density-95.
Authors: H Tochio / F Hung / M Li / D S Bredt / M Zhang /
Abstract: The second PDZ domain of postsynaptic density-95 (PSD-95 PDZ2) plays a critical role in coupling N-methyl-D-aspartate receptors to neuronal nitric oxide synthase (nNOS). In this work, the solution structure of PSD-95 PDZ2 was determined to high resolution by NMR spectroscopy. The structure of PSD-95 PDZ2 was compared in detail with that of alpha1-syntrophin PDZ domain, as the PDZ domains share similar target interaction properties. The interaction of the PSD-95 PDZ2 with a carboxyl-terminal peptide derived from a cytoplasmic protein CAPON was studied by NMR titration experiments. Complex formation between PSD-95 PDZ2 and the nNOS PDZ was modelled on the basis of the crystal structure of the alpha1-syntrophin PDZ/nNOS PDZ dimer. We found that the prolonged loop connecting the betaB and betaC strands of PSD-95 PDZ2 is likely to play a role in both the binding of the carboxyl-terminal peptide and the nNOS beta-finger. Finally, the backbone dynamics of the PSD-95 PDZ2 in the absence of bound peptide were studied using a model-free approach. The "GLGF"-loop and the loop connecting alphaB and betaF of the protein display some degree of flexibility in solution. The rest of the protein is rigid and lacks detectable slow time-scale (microseconds to milliseconds) motions. In particular, the loop connecting betaB and betaC loop adopts a well-defined, rigid structure in solution. It appears that the loop adopts a pre-aligned conformation for the PDZ domain to interact with its targets.

#3: Journal: To be Published
Title: Structure of the Third Pdz Domain of Psd-95 Protein Complexed with Kketwv Peptide Ligand
Authors: Saro, D. / Wawrzak, Z. / Martin, P. / Vickrey, J. / Paredes, A. / Kovari, L. / Spaller, M.

#4: Journal: Mol Cell / Year: 2001
Title: Structure of the SH3-guanylate kinase module from PSD-95 suggests a mechanism for regulated assembly of MAGUK scaffolding proteins.
Authors: A W McGee / S R Dakoji / O Olsen / D S Bredt / W A Lim / K E Prehoda /
Abstract: Membrane-associated guanylate kinases (MAGUKs), such as PSD-95, are modular scaffolds that organize signaling complexes at synapses and other cell junctions. MAGUKs contain PDZ domains, which recruit signaling proteins, as well as a Src homology 3 (SH3) and a guanylate kinase-like (GK) domain, implicated in scaffold oligomerization. The crystal structure of the SH3-GK module from PSD-95 reveals that these domains form an integrated unit: the SH3 fold comprises noncontiguous sequence elements divided by a hinge region and the GK domain. These elements compose two subdomains that can assemble in either an intra- or intermolecular fashion to complete the SH3 fold. We propose a model for MAGUK oligomerization in which complementary SH3 subdomains associate by 3D domain swapping. This model provides a possible mechanism for ligand regulation of oligomerization.

History

Deposition	Jul 15, 2010	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Jul 20, 2011	Provider: repository / Type: Initial release
Revision 1.1	Aug 10, 2011	Group: Database references
Revision 1.2	Aug 30, 2017	Group: Data collection / Category: em_software Item: _em_software.fitting_id / _em_software.image_processing_id
Revision 1.3	Nov 13, 2019	Group: Data collection / Other / Category: cell / Item: _cell.Z_PDB

Assembly

Deposited unit

A: DISKS LARGE HOMOLOG 4

B: DISKS LARGE HOMOLOG 4

Summary:

• 160 kDa, 2 polymers
• Cα atoms only: AB

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	160,406	2
Polymers	160,406	2
Non-polymers	0	0
Water	0

1

Summary:

• Idetical with deposited unit
• defined by author&software

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Calculated values:

Method	PISA

Components

#1: Protein

A B DISKS LARGE HOMOLOG 4 / POSTSYNAPTIC DENSITY PROTEIN 95 / PSD-95 / SYNAPSE-ASSOCIATED PROTEIN 90 / SAP-90 / SAP90 / Coordinate model: Cα atoms only

Details:

Mass: 80203.047 Da / Num. of mol.: 2 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) RATTUS NORVEGICUS (Norway rat) / Plasmid: PGEX-6P / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21 / References: UniProt: P31016

• Compound details: ENGINEERED RESIDUE IN CHAIN A, THR 9 TO ALA ENGINEERED RESIDUE IN CHAIN A, GLU 23 TO LYS ENGINEERED RESIDUE IN CHAIN A, GLN 594 TO ARG

Experimental details

Experiment

Experiment

Method
ELECTRON MICROSCOPY
SOLUTION SCATTERING

• EM experiment: Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

Sample preparation

• Component: Name: RAT PSD-95 / Type: COMPLEX
• Buffer solution: Name: 20MM TRIS/HCL, 5MM DTT, 1MM EDTA, / pH: 7.5 / Details: 20MM TRIS/HCL, 5MM DTT, 1MM EDTA,
• Specimen: Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
• EM staining: Type: NEGATIVE / Material: Uranyl Acetate
• Specimen support: Details: CARBON

Data collection

• Microscopy: Model: FEI TECNAI 10 / Details: LOW DOSE
• Electron gun: Electron source: TUNGSTEN HAIRPIN / Accelerating voltage: 100 kV / Illumination mode: FLOOD BEAM
• Electron lens: Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 43000 X / Nominal defocus max: 2250 nm / Nominal defocus min: 900 nm / Cs: 3.6 mm
• Specimen holder: Tilt angle max: 0.1 ° / Tilt angle min: 0 °
• Image recording: Film or detector model: GENERIC FILM
• Image scans: Num. digital images: 2
• Radiation wavelength: Relative weight: 1

Soln scatter

Type	ID	Buffer name	Conc. range (mg/ml)	Data reduction software list	Detector type	Mean guiner radius (nm)	Num. of time frames	Protein length	Source beamline	Source class	Source type	Temperature (K)
x-ray	1	20MM TRIS/HCL, 5MM DTT, 1MM EDTA, PH 7.5, 50MM NACL	1-8	OTOKO/GNOM	MULTIWIRE 2-D	4.36	64	14	STATION 2.1	Y	SRS	277
modelling	2

Processing

EM software

ID	Name	Category
1	UCSF Chimera	model fitting
2	EMAN	3D reconstruction

• CTF correction: Details: PARAMETERS DETERMINED USING SCATTERING CURVE
• Symmetry: Point symmetry: C₁ (asymmetric)
• 3D reconstruction: Resolution: 22.9 Å / Num. of particles: 7854 / Nominal pixel size: 3.667 Å / Actual pixel size: 3.667 Å; Details: THE COORDINATES DEPOSITED ARE FROM A COMBINED SAXS/EM STUDY. THE DOMAINS IN THE PROTEINS (HIGH RESOLUTION STRUCTURES FROM THE PDB) ARE POSITIONED RELATIVE TO ONE ANOTHER USING A SAXS CURVE, THIS COMPOSITE STRUCTURE IS THEN FITTED INTO AN EM MAP. DOMAINS PROVIDED BY X-RAY, NMR POSITIONED BY BUNCH ( PETOUKHOV, M. V. & SVERGUN, D. I. (2005). GLOBAL RIGID BODY MODELING OF MACROMOLECULAR COMPLEXES AGAINST SMALL- ANGLE SCATTERING DATA. BIOPHYS J 89, 1237-50.) REFINEMENT SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-1761. (DEPOSITION ID: 7384).; Symmetry type: POINT
• Atomic model building: Protocol: RIGID BODY FIT / Space: REAL; Details: METHOD--A MAP WAS GENERATED FROM THE SAXS MODEL COORDINATES AT A RESOLUTION MATCHING THE EXPERIMENTAL MAP. T HIS CALCULATED MAP WAS FITTED INTO THE EXPERIMENTAL MAP BY MAXIMIZING THE CROSS-CORRELATION WITH THE EXPERIMENTAL MAP. THE COORDINATES WERE THEN REPLACED IN THE CALCULATED MAP TO GENERATE THE FINAL ENTRY. REFINEMENT PROTOCOL--DOCKED USING CHIMERA

Atomic model building

ID	PDB-ID	3D fitting-ID
1	1IU0 1iu0	1
2	1QLC 1qlc	1
3	1TP5 1tp5	1
4	1KJW 1kjw	1

• Refinement: Highest resolution: 22.9 Å

Refinement step

Cycle: LAST / Highest resolution: 22.9 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	1439	0	0	0	1439

• Soln scatter model: Method: RIGID BODY MODELLING; Conformer selection criteria: TWO MAJOR CONFORMERS WERE PRESENT IN THE POOL OF REFINED SAXS MODELS CHAIN A REPRESENTS THE BEST AGREEMENT WITH THE EXPERIMENTAL SAXS DATA (CHI=3.3). CHAIN B IS REPRESENTATIVE OF THE SECOND CONFORMATION (CHI=3.8).; Details: NUMBER OF TIME FRAMES USED 24 (60S, 4.25M CAMERA), 40(60S, 1M CAMERA). PROTEIN CONCENTRATION 1 MG/ML (4.25M CAMERA) 8 MG/ML (1M CAMERA); Entry fitting list: PROGRAM PRE-BUNCH 1IU0/1QLC/1TP5/1KJW + SEQUENCE DATA; Num. of conformers calculated: 20 / Num. of conformers submitted: 2 / Software author list: PETOUKHOV, M. V. & SVERGUN, D. I. / Software list: SASREF7/BUNCH8