Microcrystal Growth Protocols for SARS-CoV-2 Mpro

M^pro microcrystals were grown using seeded batch crystallization in the XBI laboratories of the European XFEL²⁷ . Seed crystals were grown from protein purified in the presence of 0.5 mM TCEP, using a sitting drop geometry by combining 250 nL M^pro protein solution (6.25 mg/ml) and 250 nL precipitant (25% PEG1500, 0.1 M MIB buffer pH 7.5, 5% DMSO), as reported previously^{28 (link)}. A seed stock was produced by adding the resulting M^pro crystals to a reaction tube containing a glass bead (Beads-for-Seeds, Jena Bioscience) and vortexing periodically for 5 s with subsequent incubation at room temperature. For the microcrystal batch crystallization, 250 μL glass seed beads were added to a 1.5 mL reaction tube, which was then filled with 900 μL precipitant solution (25% PEG1500, 0.1 M MIB buffer pH 7.5, 5% DMSO) mixed with 100 μL seed stock and 100 μL M^pro protein solution (35 mg/ml). Subsequently, crystals were grown in a shaker at 18 °C at 900 rpm overnight. The reduced and oxidized forms were crystallized separately. Resulting crystals were thin plates with a size ranging from 3–15 μm. Crystal concentration was adjusted by allowing the crystals to settle overnight and removing supernatant accordingly. Final crystal slurry was filtered through a 30 μm mesh gravity filter (Sysmex CellTrics) before injection.
Protein crystals for single-crystal rotation experiments were produced as previously reported^{28 (link)}, using orthorhombic seeds and reduced protein at 6.25 mg/mL. For the ligand free and S-calpeptin containing crystallization experiments, the same reduced protein batch was used. The S-calpeptin compound was dried in the well prior to crystallization mixture addition, yielding a maximum concentration of 5 mM.