Before a crystal can be flash-cooled in liquid nitrogen for transport and data collection, it must be briefly transferred to a cryoprotectant solution containing glycerol, ethylene glycol, or a high-concentration PEG. The goal is vitrification: replacing the solvent in the crystal with a glass-like solid that avoids forming crystalline ice during cooling. Crystalline ice results in poor diffraction quality. The challenge is that the cryoprotectant must not disrupt protein packing, and both concentration and soaking time must be tested empirically for each crystal system. For fragile crystals, stepwise transfer into increasing cryoprotectant concentrations is safer than a direct jump to full concentration. The crystal is inspected after each transfer step; cracking, opacity, or dissolution indicates an incompatible cryoprotectant or an excessive concentration. Once a cryosolution tolerated by the crystal is found, excess liquid is removed from around the mounted loop, and the crystal is immediately plunged into liquid nitrogen. At the synchrotron, data collection is relatively fast, often minutes per crystal. As mentioned above, dataset quality largely depends on the earlier stages: the crystal’s quality (internal order) and the quality of the cryoprotection. Direct access to a high-brilliance synchrotron source, like MAX IV Laboratory in Lund, is a clear advantage because it removes the need to send crystals to remote synchrotrons. This is also essential for crystallographic fragment screening, as in this case a large number of crystals are grown directly at the beamline.
