Microcrystallization techniques for serial femtosecond crystallography using photosystem II from Thermosynechococcus elongatus as a model system.

Serial femtosecond crystallography (SFX) is a new emerging method, where X-ray diffraction data are collected from a fully hydrated stream of nano-or microcrystals of biomolecules in their mother liquor using high-energy, X-ray free-electron lasers. The success of SFX experiments strongly depends on the ability to grow large amounts of well-ordered nano/microcrystals of homogeneous size distribution. While methods to grow large single crystals have been extensively explored in the past, method developments to grow nano/microcrystals in sufficient amounts for SFX experiments are still in their infancy. Here, we describe and compare three methods (batch, free interface diffusion (FID) and FID centrifugation) for growth of nano/microcrystals for time-resolved SFX experiments using the large membrane protein complex photosystem II as a model system.