The initial crystallization conditions identified during screening almost always require further optimization. Well-formed crystals typically range from 0.5 to 2 mm in size, depending on the protein and crystallization conditions. During optimization, parameters such as buffer pH, salt concentration, additive concentration, and even the precipitant type may need adjustment to produce crystals of the required size. We may also need to adjust drop volume to optimize crystal size and explore alternative temperatures during this stage. Volume and temperature variations may potentially affect crystal nucleation and growth rates, improving crystal quality and yield (e.g., for crystallographic fragment screening). We use automated, temperature-controlled plate hotels designed for high-throughput protein crystallization and monitoring. We monitor crystallization plates using an imaging system, such as the Formulatrix Rock Imager. Crystallization of protein-ligand complexes is extensively used in structure-based drug design (SBDD). Co-crystallization with a ligand of interest often begins after establishing crystallization conditions for the apo-protein. There are two primary methods for obtaining a ligand complex: (1) adding a ligand solution to the crystallization drop containing crystals and soaking the crystals with the ligand, and (2) co-crystallization with the ligand. Soaking may work because protein crystals contain a relatively high percentage of solvent (crystallization buffer), allowing the ligand to diffuse into the crystal. If the ligand-binding site within the crystal is exposed and accessible to solvent, the ligand will bind. Conversely, if the site is deeply buried within the protein or obscured by crystal contacts, it becomes inaccessible to ligands. In many cases, ligand soaking cracks the crystal, resulting in poor X-ray diffraction. In such cases, we use co-crystallization with a ligand, which can be applied in different ways. The simplest approach is to mix the ligand solution with the protein before preparing crystallization drops. However, ligand binding may trigger a conformational transition in the protein, potentially requiring reoptimization of crystallization conditions. In some cases, co-crystallization may be combined with seeding, which may help in obtaining crystals of the complex. We also need to note that, in all these experiments, ligand affinity is an important factor to consider. More information, with detailed discussion and guidelines for the successful generation of protein–ligand complex crystals, can be found in this excellent publication. You can also find some practical details on the Hampton Research website.
