Accurate and Efficient SAXS/SANS Implementation Including Solvation Layer Effects Suitable for Molecular Simulations.

Ballabio F, Paissoni C, Bollati M, de Rosa M, Capelli R, Camilloni C, J Chem Theory Comput (2023) Europe PMC

SASDSN7 – Full length human mature gelsolin in closed state

MWexperimental 85 kDa
MWexpected 85 kDa
VPorod 128 nm3
log I(s) 5.75×10-2 5.75×10-3 5.75×10-4 5.75×10-5
Gelsolin small angle scattering data  s, nm-1
ln I(s)
Gelsolin Guinier plot ln 5.75×10-2 Rg: 3.2 nm 0 (3.2 nm)-2 s2
Gelsolin Kratky plot 1.104 0 3 sRg
Gelsolin pair distance distribution function Rg: 3.2 nm 0 Dmax: 11.1 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Gelsolin GROMACS model

Synchrotron SAXS data from solutions of gelsolin in 20 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.4 were collected on the B21 beam line at the Diamond Light Source (Didcot, UK) using a Eiger 4M detector at a sample-detector distance of 3.5 m and at a wavelength of λ = 0.1 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 2.01 mg/ml was measured at 25°C. 20 successive 1 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.

Human plasma isoform of GSN devoid of the signal peptide (mature form) was produced in E. coli SHuffle® cells (New England Biolabs) upon addition of 0.5 mM IPTG and incubation for 16 hours at 18 °C. Cells were lysed in a Basic Z Bench top (Constant Systems Limited, U.K.) at 25 kPSI, and the clarified extract passed through a HisTrap HP column (all chromatographic media from GE-Healthcare). Further polishing was obtained by anion-exchange (Resource Q) followed by size-exclusion chromatography (HiLoad 16/600 Superdex 200).

Gelsolin (GSN)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   85.4 kDa
UniProt   P06396 (28-782)
Sequence   FASTA