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

SASDWE3 – Cyanobacterial circadian clock protein KaiB-KaiC complex (71% deuterated-KaiB and hydrogenated-KaiC in 100% v/v D₂O ) measured by SEC-SANS

Circadian clock oscillator protein KaiB
Circadian clock oscillator protein KaiC
MWI(0) 333 kDa
MWexpected 427 kDa
log I(s) 5.46×10-1 5.46×10-2 5.46×10-3 5.46×10-4
Circadian clock oscillator protein KaiB Circadian clock oscillator protein KaiC small angle scattering data  s, nm-1
ln I(s)
Circadian clock oscillator protein KaiB Circadian clock oscillator protein KaiC Guinier plot ln 5.47×10-1 Rg: 4.3 nm 0 (4.3 nm)-2 s2
(sRg)2I(s)/I(0)
Circadian clock oscillator protein KaiB Circadian clock oscillator protein KaiC Kratky plot 1.104 0 3 sRg
p(r)
Circadian clock oscillator protein KaiB Circadian clock oscillator protein KaiC pair distance distribution function Rg: 4.1 nm 0 Dmax: 11.7 nm

Data validation


There are no models related to this curve.

SANS data from solutions of the Cyanobacterial circadian clock protein KaiB-KaiC complex (71% deuterated-KaiB and 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 and 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 6.8 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 KaiB (kaiB)
Mol. type   Protein
Organism   Synechococcus sp. (strain ATCC 27144 / PCC 6301 / SAUG 1402/1)
Olig. state   Hexamer
Mon. MW   11.8 kDa
 
UniProt   Q5N595 (1-102)
Sequence   FASTA
 
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