Advances in the nanostructure characterization of biological hydrogels formed by the prion-like domain of EARLY FLOWERING 3.

Hydrogels exhibit traits of both liquids and solids, forming a 3D structural network with generally high water content, high porosity and increasing stability over time. However, probing the structure of hydrogels is challenging due to the semi-disordered nature of their internal molecular organization and the presence of multiple species with varying dynamics. Small-angle X-ray scattering (SAXS) is well suited to study materials of this nature due to its ability to probe different length scales and to provide information as to the structure and conformation of different molecular species within the hydrogel. Here, we used the prion-like domain from the plant thermosensory protein EARLY FLOWERING 3 (ELF3) as a model hydrogel for structural characterization by SAXS. To overcome sample-handling challenges, we developed an X-ray- and vacuum-compatible sample cell for hydrogel manipulation that allows direct measurement in the X-ray beam. Furthermore, by using a three-component model, which gathers the scattering contributions from the different species in the hydrogel, which arise from individual and aggregated oligomers as well as from their stacking into a loose lamellar phase, we were able to satisfactorily fit the experimental data and provide a full description of the nanostructure of the ELF3 hydrogel.