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Synchrotron SAXS data from solutions of pseudoamylase BAM9 bound to alpha-amylase AMY3 in 20 mM HEPES, 100 mM NaCl, 0.2 mM TCEP, pH 7 were collected on the 12.3.1 (SIBYLS) beam line at the Advanced Light Source (ALS; Berkeley, CA, USA) using a Pilatus3 X 2M detector at a sample-detector distance of 2.1 m and at a wavelength of λ = 0.1027 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A sample at 4 mg/ml was injected onto a Shodex KW-803 column at 10°C. 20 successive 2 second frames were collected through the sample elution peak. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.
Purified protein samples of AMY3 and BAM9 combined were shipped overnight at 4°C and analyzed using SEC-SAXS with in-line with UV/vis absorbance at 280nm and Multi-angle light scattering (MALS). Radially averaged SAXS data files were processed and analyzed in Scatter IV and RAW (Hopkins, 2024).
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s, nm-1
