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

SASDWD3 – Cyanobacterial circadian clock protein KaiB-KaiC complex (hydrogenated-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) 442 kDa
MWexpected 427 kDa
log I(s) 5.82×10-1 5.82×10-2 5.82×10-3 5.82×10-4
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 5.82×10-1 Rg: 4.9 nm 0 (4.9 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: 4.4 nm 0 Dmax: 14.4 nm

Data validation


There are no models related to this curve.

SANS data from solutions of the Cyanobacterial circadian clock protein KaiB-KaiC complex (hydrogenated-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 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. 12 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