Fixed-target pump-probe SFX: eliminating the scourge of light contamination.

X-ray free-electron laser (XFEL) light sources have enabled the rapid growth of time-resolved structural experiments, which provide crucial information on the function of macromolecules and their mechanisms. Here, the aim was to commission the SwissMX fixed-target sample-delivery system at the SwissFEL Cristallina experimental station using the PSI-developed micro-structured polymer (MISP) chip for pump-probe time-resolved experiments. To characterize the system, crystals of the light-sensitive protein light-oxygen-voltage domain 1 (LOV1) from Chlamydomonas reinhardtii were used. Using different experimental settings, the accidental illumination, referred to as light contamination, of crystals mounted in wells adjacent to those illuminated by the pump laser was examined. It was crucial to control the light scattering from and through the solid supports otherwise significant contamination occurred. However, the results here show that the opaque MISP chips are suitable for defined pump-probe studies of a light-sensitive protein. The experiment also probed the sub-millisecond structural dynamics of LOV1 and indicated that at Δt = 10 µs a covalent thioether bond is established between reactive Cys57 and its flavin mononucleotide cofactor. This experiment validates the crystals to be suitable for in-depth follow-up studies of this still poorly understood signal-transduction mechanism. Importantly, the fixed-target delivery system also permitted a tenfold reduction in protein sample consumption compared with the more common high-viscosity extrusion-based delivery system. This development creates the prospect of an increase in XFEL project throughput for the field.