Small-angle neutron scattering studies on the AMPA receptor GluA2 in the resting, AMPA-bound and GYKI-53655-bound states.

Larsen AH, Dorosz J, Thorsen TS, Johansen NT, Darwish T, Midtgaard SR, Arleth L, Kastrup JS, IUCrJ 5(Pt 6):780-793 (2018) Europe PMC

SASDD36 – AMPA subtype ionotropic Glutamate receptor GluA2 in the GYKI-53655 bound state, in stealth DDM detergents

Glutamate receptor 2
MWexperimental 319 kDa
MWexpected 368 kDa
VPorod 384 nm3
log I(s) 1.10×10-1 1.10×10-2 1.10×10-3 1.10×10-4
Glutamate receptor 2 small angle scattering data  s, nm-1
ln I(s)
Glutamate receptor 2 Guinier plot ln 1.10×10-1 Rg: 6.3 nm 0 (6.3 nm)-2 s2
(sRg)2I(s)/I(0)
Glutamate receptor 2 Kratky plot 1.104 0 3 sRg
p(r)
Glutamate receptor 2 pair distance distribution function Rg: 6.2 nm 0 Dmax: 18.6 nm

Data validation


Fits and models


log I(s)
 s, nm-1

SANS data from solutions of Glutamate receptor 2 (GluA2) in the GYKI-53655 bound state in D2O based buffer. 1 mM GYKI-53655, 20 mM Tris/DCl, 100 mM NaCl, 0.5 mM deuterated n-dodecyl-β-D-maltopyranoside (synthesized to match out at 100% D2O), pH 7.5 were collected on the KWS1 camera at the FRM2 storage ring (Munich, Germany) using a 6Li-Scintillator 1 mm thickness + photomultiplier detector at a wavelength of λ = 0.5 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 0.31 mg/ml was measured at 10°C. 15 successive 900 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Sample of GluA2 in the GYKI-53655 bound state. The SANS data were fitted with a mixture of GluA2 in the tetrameric GYKI-53655 bound state (pdb-code 5l1h) and a fraction of oligomers of the tetramer (best fit: fit85.dat). The oligomers were described with a fractal structure factor (Teixeira, J. (1988). J. Appl. Crystallogr. 21, 781-785). Data were fitted with WillItFit (Pedersen, M. C., Arleth, L. & Mortensen, K. (2013). J. Appl. Crystallogr. 46, 1894-1898). The SANS data were measured in three settings (sample/collimation): 1.5m/4m, 4m/4m, and 8m/8m. The attached data are merged into one data set with all three settings. Pair distance distribution functions were calculated with BayesApp (www.bayesapp.org). Due to relatively low protein concentration, the concentration measurement was inaccurate, and the MW was therefore evaluated by (concentration independent) Porod analysis using the Porod volume calculator implemented in PRIMUS, and with a volume-to-mass conversion constant of 0.83 kDa/nm^3 (Gekko, K. & Noguchi, H. (1979). J. Phys. Chem. 83, 2706-2714; Squire, P. G. & Himmel, M. E. (1979). Arch. Biochem. Biophys. 196, 165-177).

Glutamate receptor 2 (GluA2)
Mol. type   Protein
Organism   Rattus norvegicus
Olig. state   Monomer
Mon. MW   367.7 kDa
 
UniProt   P19491
Sequence   FASTA