Standard Sample Preparation for Serial Femtosecond Crystallography

The development of serial crystallography (SX), including serial synchrotron crystallography (SSX) at synchrotron sources and serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs), has facilitated the collection of high-resolution diffraction data from micron-sized crystals, providing unique insights into the structures and dynamics of biomolecules at room temperature. Standard samples are essential for the commissioning of new XFEL instruments and the validation of experimental setups. In this review, we summarize currently used standard proteins and describe representative microcrystal preparation workflows for four widely adopted models, lysozyme, myoglobin, iq-mEmerald, and photoactive yellow protein (PYP), drawing on established methodologies and accumulated experience from their applications at the European XFEL. By consolidating existing knowledge and integrating protocols that have been systematically refined and optimized through our experimental efforts, this review aims to provide practical guidance for the serial crystallography community, thereby enhancing reproducibility and ensuring consistent experimental performance across facilities.