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

Abstract

A monodispersed and ideal solution is a key requirement of BioSAXS to allow one to extract structural information from the recorded pattern. On-line size exclusion chromatography (SEC) marked a major breakthrough, separating particles present in solution according to their size. Scattering curves with identical shape under an elution peak can be averaged and further processed free from contamination. However, this is not always straightforward, separation is often incomplete. Software have been developed to deconvolve the contributions from the different species (molecules or oligomeric forms) within the sample. In this chapter, we present the general workflow of a SEC-SAXS experiment. We present recent instrumental and data analysis developments that have improved the quality of recorded data, extended the potential of SEC-SAXS and turned it into a mainstream approach. We report a comparative analysis of two macromolecular systems using various deconvolution approaches that have been developed over the last years. Parallel analysis appears to be the best cross-validation method to assess the reliability of the reconstructed isolated species patterns that can safely be used as a support for meaningful molecular modeling.