Structure-based screening of binding affinities via small-angle X-ray scattering

Chen P, Masiewicz P, Perez K, Hennig J, (2019) DOI

SASDFJ8 – Glutamate/aspartate import solute-binding protein (DEBP) in the presence of glutamate - Glu-bound 10-fold excess

Glutamate/aspartate import solute-binding protein
MWexperimental 31 kDa
MWexpected 32 kDa
VPorod 39 nm3
log I(s) 6.70×101 6.70×100 6.70×10-1 6.70×10-2
Glutamate/aspartate import solute-binding protein small angle scattering data  s, nm-1
ln I(s)
Glutamate/aspartate import solute-binding protein Guinier plot ln 6.71×101 Rg: 2.1 nm 0 (2.1 nm)-2 s2
(sRg)2I(s)/I(0)
Glutamate/aspartate import solute-binding protein Kratky plot 1.104 0 3 sRg
p(r)
Glutamate/aspartate import solute-binding protein pair distance distribution function Rg: 2.1 nm 0 Dmax: 6.4 nm

Data validation


There are no models related to this curve.

Synchrotron SAXS data from solutions of DEBP with a 10-fold excess of glutamate in 100 mM NaCl, 20 mM NaPO4, 0.5 mM TCEP, pH 7.4 were collected on the BM29 beam line at the ESRF (Grenoble, France) using a Pilatus 1M detector at a sample-detector distance of 2.8 m and at a wavelength of λ = 0.09919 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 2.20 mg/ml was measured at 20°C. 12 successive 2 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.

Glu-bound DEBP at a ligand:protein ratio of 10:1. Ligand condition is prepared by dissolving glutamate in powder form (Sigma-Aldrich) directly into the SAXS buffer, followed by dilution to the target concentration with additional SAXS buffer. Note that the scattering intensity therefore contains Na+ counterions that was not directly subtracted.

Glutamate/aspartate import solute-binding protein
Mol. type   Protein
Organism   Escherichia coli
Olig. state   Monomer
Mon. MW   32.1 kDa
 
UniProt   P37902 (28-302)
Sequence   FASTA