In Situ Soft X-Ray Characterization of in vivo Engineered Fluorescent Protein Liquid-Liquid Phase Separation and Crystallization

The aim of the study is to probe three-dimensional structural information at the meso scale to characterize in vivo phase transition of a heterologously expressed protein in a mammalian cell expression system using correlative fluorescence/cryo-soft X-ray tomography. The first objective is to characterize the structural features of the new type of sorbitol induced intracellular XPA crystals and probe the possible involvement of membrane structures to nucleate these structures by trapping the early nucleation events of these crystalline plates thanks to the high number of tomograms that can be recorded in cryoSXT beamtime sessions. The second objective is to describe the dendrite like XPA precipitate observed in close interaction with membrane as seen by fluorescence microscopy. The third objective is to probe if the widespread inhomogeneous distribution of fluorescent XPA within the spinodal like phase, in which the carbon density could be intermediate between the dilute and crystalline dense phase, can be probed with cryoSXT. In other terms, to see if the quantitative carbon absorption levels measured with cryoSXT (measuring the X-ray linear absorption coefficient – LAC) correlates with the fluorescence pattern measured by cryoSIM.