Ligand Screening and Discovery using Cocktail Soaking and Automated MicroED.

Cocktail soaking using single-crystal X-ray diffraction (SC-XRD) has previously allowed high-throughput crystallographic screening of ligands against protein targets. However, protein microcrystals are not amenable to this approach if they are too small to yield strong diffraction patterns. In this study, we developed a workflow integrating cocktail soaking with automated microcrystal electron diffraction (MicroED) to allow rapid ligand screening, structure determination, and binding analysis directly from microcrystals. This can improve the successful hit rate, because binding is often more efficient when smaller crystals are soaked in the ligand. The approach was validated with known ligands of thermolysin and identified novel binding interactions for ligands of proteinase K. The structures of multiple protein-ligand complexes, including ligands with weak binding affinities, could be solved quickly. Their estimated relative binding affinities are in good agreement with previous work and independent microscale thermophoresis (MST) measurements.