SEC-SAXS: experimental set-up and software developments build up a powerful tool.

Aurélien Thureau.

SASDPH3 – Benchmarking extraction methods of scattering patterns of poorly resolved components from a SEC-SAXS dataset of BSA.

Bovine Serum Albumin
MWexperimental 220 kDa
MWexpected 199 kDa
VPorod 370 nm3
log I(s) 9.85×10-1 9.85×10-2 9.85×10-3 9.85×10-4
Bovine Serum Albumin small angle scattering data  s, nm-1
ln I(s)
Bovine Serum Albumin Guinier plot ln 9.85×10-1 Rg: 5.2 nm 0 (5.2 nm)-2 s2
Bovine Serum Albumin Kratky plot 1.104 0 3 sRg
Bovine Serum Albumin pair distance distribution function Rg: 5.3 nm 0 Dmax: 17.6 nm

Data validation

There are no models related to this curve.

X-ray synchrotron radiation scattering data, I(s) vs s, (s = 4πsinθ/λ, and 2θ is the scattering angle) were collected from a sample of bovine serum albumin using continuous-flow size-exclusion chromatography SAXS (SEC-SAXS) at the SWING beamline (wavelength, λ: 0.1033 nm) on the Synchrotron SOLEIL (Gif sur Yvette, France). The BSA sample (Sigma-Aldrich A7906) was dissolved in Hepes 20mM pH7.4 NaCl 150mM. The solution was stored at 5°C for a few days before measurements, conditions that allow for a well-documented self-association of the protein into dimers, trimers and higher order oligomers. 50 µL of 10.8 mg/mL solution were loaded onto a BioResolve SEC column 200 Å, 2.5 µm, 4.6 x 300 mm (from WatersTM) at a flow rate of 0.3 ml/min. A total of 720 x 0.99 second SAXS data frames were recorded throughout the BSA elution (180 before void volume and 540 after void volume) . After background solvent corrections, the 250 first frames after the void volume were analyzed. SVD analysis performed in US-SOMO or RAW suggests that four species are necessary to account for the complete dataset. Three different methods (EFA, REGALS, US-SOMO) were tested to deconvolute the various species. The presented SAXS curve is the peak3 of the US-SOMO analysis corresponding to the trimer of BSA according to the Molecular Weight. The 250 subtracted frames from the SEC-SAXS measurements and the results of the three methods are available in the zip file.

Bovine Serum Albumin (BSA)
Mol. type   Protein
Organism   Bos taurus
Olig. state   Trimer
Mon. MW   66.4 kDa
UniProt   P02769 (25-607)
Sequence   FASTA