Global fitting of multiple data frames from SEC–SAXS to investigate the structure of next-generation nanodiscs

Abstract

The combination of online size-exclusion chromatography and small-angle X-ray scattering (SEC–SAXS) is rapidly becoming a key technique for structural investigations of elaborate biophysical samples in solution. Here, a novel model-refinement strategy centred around the technique is outlined and its utility is demonstrated by analysing data series from several SEC–SAXS experiments on phospholipid bilayer nanodiscs. Using this method, a single model was globally refined against many frames from the same data series, thereby capturing the frame-to-frame tendencies of the irradiated sample. These are compared with models refined in the traditional manner, in which refinement is based on the average profile of a set of consecutive frames from the same data series without an in-depth comparison of individual frames. This is considered to be an attractive model-refinement scheme as it considerably lowers the total number of parameters refined from the data series, produces tendencies that are automatically consistent between frames, and utilizes a considerably larger portion of the recorded data than is often performed in such experiments. Additionally, a method is outlined for correcting a measured UV absorption signal by accounting for potential peak broadening by the experimental setup.

A retrospective look at developments in size exclusion chromatography from the early Hamielec research era to the present day

“…Separations are not perfect and so deconvolution methods are currently recommended for data analysis. [66,67] There are great possibilities for further exploitation of the technique in biomedical research for probing 3-D shapes and conformations of biomolecules in solution. Researchers in the field look ahead to an expanded use of SAXS in the coming years to drive advances in structural biology and to investigate various cellular structures.…”