Size-exclusion chromatography–small-angle neutron scattering system optimized for an instrument with medium neutron flux

Morishima K, Inoue R, Nakagawa T, Shimizu M, Sakamoto R, Oda T, Mayumi K, Sugiyama M, Journal of Applied Crystallography 58(2) (2025) DOI

SASDWC3 – Apoferritin, from SEC-SANS

Ferritin light chain
MWI(0) 399 kDa
MWexpected 476 kDa
log I(s) 9.41×10-1 9.41×10-2 9.41×10-3 9.41×10-4
Ferritin light chain small angle scattering data  s, nm-1
ln I(s)
Ferritin light chain Guinier plot ln 9.41×10-1 Rg: 5.4 nm 0 (5.4 nm)-2 s2
(sRg)2I(s)/I(0)
Ferritin light chain Kratky plot 1.104 0 √3 sRg
p(r)
Ferritin light chain pair distance distribution function Rg: 5.4 nm 0 Dmax: 23.1 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Ferritin light chain PDB (PROTEIN DATA BANK) model
SANS data from solutions of apoferritin in 100 mM Tris-HCl, 100 mM NaCl in 100% v/v D₂O, pH 7.5 were collected on the SANS-U beam line at JRR-3 (Japan Atomic Energy Agency; Ibaraki, Japan) using a ORDELA, model 2660N detector at a sample-detector distance of 4 and 1.03 m at a wavelength of λ = 0.6 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Stop-flow mode in-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 500.00 μl sample at 12 mg/ml was injected onto a Cytiva Superdex 200 Increase 10/300 column at 25°C. 24 successive 600 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.

SEC elution paused during SANS measurements.

Ferritin light chain
Mol. type   Protein
Organism   Equus caballus
Olig. state   24-mer
Mon. MW   19.8 kDa
 
UniProt   P02791 (2-175)
Sequence   FASTA
 
PDB ID   4V1W