Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument

Bucciarelli S, Midtgaard S, Nors Pedersen M, Skou S, Arleth L, Vestergaard B, Journal of Applied Crystallography 51(6) (2018) DOI

SASDE25 – Human insulin hexamer from in-house SEC-SAXS

Human insulin
MWexperimental 29 kDa
MWexpected 35 kDa
VPorod 39 nm3
log I(s) 5.30×10-2 5.30×10-3 5.30×10-4 5.30×10-5
Human insulin small angle scattering data  s, nm-1
ln I(s)
Human insulin Guinier plot ln 5.30×10-2 Rg: 2 nm 0 (2 nm)-2 s2
(sRg)2I(s)/I(0)
Human insulin Kratky plot 1.104 0 3 sRg
p(r)
Human insulin pair distance distribution function Rg: 1.9 nm 0 Dmax: 5.9 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Human insulin DAMFILT model

log I(s)
 s, nm-1
Human insulin PDB model

SAXS data from solutions of Human insulin hexamer from in-house SEC-SAXS in 7 mM sodium phosphate, 60 mM phenol, 200 µM Zn(CH3COO)2, 23 mM NaCl, pH 7.4 were collected on a Xenocs BioXolver L instrument at the Copenhagen University, Department of Drug Design and Pharmacology (Copenhagen, Denmark) using a 20Hz Pilatus 300K detector at a sample-detector distance of 0.7 m and at a wavelength of λ = 0.134 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle).

The HI sample (500 microlitres at 3.5 mg/ml) was injected onto a GE Healthcare S200 Increase 10/300 (24 ml) column at a flow rate of 0.1 ml/min at 23°C. A total of 120 x 30 second SAXS data frames were recorded throughout the HI elution. After background solvent corrections, SEC-SAXS data frames 71-83, corresponding to hexameric HI, were scaled, averaged and binned logarithmically to produce the data displayed in this entry. Additional files, including plots of the selected frames are included in the additional files of the full entry zip archive.

Human insulin (HI)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Hexamer
Mon. MW   5.8 kDa
 
UniProt   P01308 (25-54 , 90-110)
Sequence   FASTA
 
PDB code   1EV3