Arizona State University

Technique development of sample delivery for serial femtosecond crystallography (SFX) using a x-ray free electron laser (X-FEL) provides the prospect of smaller sample volumes and the ability for fast mixing in the liquid jet. Because samples are not immobilized or frozen in SFX, determination of conformational changes in space and time by the induction of conformational changes “on the fly” is possible. Fast mixing of two liquids, such as enzyme and substrate, immediately before being exposed to the x-ray beam would allow conformational changes to be seen by snapshot diffraction. As a model for the liquid mixing jet, mKate, a protein that undergoes a pH dependent conformational change was chosen. Crystallization conditions to produce mKate microcrystals have been established but the time scale of the mKate conformational change is unknown making a mixing time difficult to estimate. To mimic the highly desirable viscous properties of lipidic cubic phase (LCP), agarose is currently being developed to deliver soluble crystals in a slow moving jet that decreases sample volume. An agarose jet with phycocyanin crystals embedded was tested at an LCLS. The agarose jet produced agarose crystals most likely caused by dehydration due to sample delivery in vacuum. In the future, agarose testing will be conducted in a helium atmosphere to prevent crystal formation. This work aims to further develop SFX by use of mixing-induced time resolved measurements for time resolved work, and an inert, viscous media that would allow for sample limited protein crystals to be probed by SFX.