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.
Keywords: model refinement; phospholipid nanodiscs; size-exclusion chromatography; small-angle scattering.
open access.