Absolute scattering length density profile of liposome bilayers obtained by SAXS combined with GIXOS - a tool to determine model biomembrane structure

Lipid membranes play an essential role in biology, acting as host matrices for biomolecules like proteins and facilitating their functions. Their structures, and structural responses to physiologically relevant interactions, i.e. with membrane proteins, provide key information for understanding biophysical mechanisms. Hence, there is a crucial need of methods to understand the effects of membrane host molecules on the lipid bilayer structure. Here, we present a purely experimental method for obtaining the absolute scattering length density (SLD) profile and the area per lipid of liposomal bilayers, by aiding the analysis of small angle X-ray scattering (SAXS) data with the volume of bare headgroups obtained from fast (20-120s) grazing incidence off-specular scattering (GIXOS) data from monolayers of the same model membrane lipid composition. The GIXOS data experimentally demonstrate that the variation of the bare headgroup volume upon lipid packing density change is small enough to allow its usage as a reference value without knowing the lipid packing stage in a bilayer. This approach also bares the advantage that the reference volume is obtained at the same aqueous environment as used for the model membrane bilayers. We demonstrate the validity of this method using several typical membrane compositions, as well as one example of a phospholipid membrane with an incorporated transmembrane peptide. This methodology allows to obtain absolute scale values rather than relative scale by using solely X-ray-based instrumentation, retaining a similar resolution of SAXS experiments. The presented method has high potential to understand structural effects of membrane proteins on the biomembrane structure. ### Competing Interest Statement The authors have declared no competing interest. * Abbreviations: (SLD) : Scattering length density (SAXS) : small X-ray scattering (GIXOS) : grazing incidence off-specular scattering (E) : envelope protein (SARS-CoV-2) : the severe acute respiratory syndrome coronavirus 2 (CGP) : carbonyl-glycerolphosphate (TER) : terminal groups (CHOL) : choline (SER) : serine