Determination of the GH3.12 protein conformation through HPLC-integrated SAXS measurements combined with X-ray crystallography

Combined small-angle X-ray scattering (SAXS) and crystallographic data were used to probe the two-step catalytic mechanism of the acyl acid-amido synthetase GH3.12. On-line size-exclusion chromatography directly upstream of the X-ray beam improved the sample homogeneity and allowed the determination of changes in the protein conformation in solution. X-ray models were fitted to the SAXS data to unambiguously determine the protein conformation in the presence of different ligands.