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

SASDWF3 – Circadian clock protein KaiC (hydrogenated-KaiC in 100% v/v D₂O ) measured by SEC-SANS

Circadian clock oscillator protein KaiC
MWI(0) 334 kDa
MWexpected 356 kDa
log I(s) 3.93×10-1 3.93×10-2 3.93×10-3 3.93×10-4
Circadian clock oscillator protein KaiC small angle scattering data  s, nm-1
ln I(s)
Circadian clock oscillator protein KaiC Guinier plot ln 3.93×10-1 Rg: 4.4 nm 0 (4.4 nm)-2 s2
(sRg)2I(s)/I(0)
Circadian clock oscillator protein KaiC Kratky plot 1.104 0 3 sRg
p(r)
Circadian clock oscillator protein KaiC pair distance distribution function Rg: 4.1 nm 0 Dmax: 11.9 nm

Data validation


There are no models related to this curve.

SANS data from solutions of the Circadian clock protein KaiC (hydrogenated-KaiC) in 50 mM sodium phosphate buffer, 150 mM NaCl, 5 mM MgCl₂, 0.5 mM EDTA, 1 mM ATP, 1 mM DTT, 50 mM arginine, 50 mM glutamine, 100% v/v D₂O, pH 7.8 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 0.50 μl sample at 5.6 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.

Circadian clock oscillator protein KaiC (kaiC)
Mol. type   Protein
Organism   Synechococcus elongatus (strain ATCC 33912 / PCC 7942 / FACHB-805)
Olig. state   Hexamer
Mon. MW   59.3 kDa
 
UniProt   Q79PF4 (2-519)
Sequence   FASTA