2.7. Crystal soaking and co-crystallisation with natural ligands and fragment hits

To obtain ligand-bound structures, soaking was performed in the crystallisation conditions described above for each protein using the hanging drop vapour diffusion method.

For ArgB, 1.5 µL of protein storage buffer containing 20 mM of ligand was mixed with 1.5 µL of reservoir solution, and drops were left to equilibrate against 500 µL of reservoir solution for 3 days. Crystals were then transferred to the drops and incubated for 16 h. A cryogenic solution was prepared by adding ethylene glycol up to 27.5% (v/v) to the mother liquor. Crystals were briefly transferred to this solution, flash-frozen in liquid N₂, and stored for data collection. To obtain an ArgB-NAG complex, co-crystallisation with 2 mM NAG was performed instead. Crystals for ArgB-NAG complex were obtained in Wizard Classic 1&2 screen (Rigaku), solution B6, and were flash-frozen in liquid N₂ after a brief soak in a solution containing mother liquor and 27.5% ethylene glycol.

ArgC crystals grown in pH 5.5 were first soaked in 1.5 µL drops containing the mother liquor and 5 mM NADP⁺ for 2 h in hanging drops that were equilibrated against a reservoir of 500 µL. Thereafter, the crystals were transferred to drops containing the crystallisation condition and an SPR-validated fragments (20 mM, 10% DMSO), which were equilibrated against 500 µL of mother liquor also containing a corresponding percentage of DMSO overnight at 19 °C. ArgC crystals grown in pH 7 were soaked with 5 mM NADP⁺ only for 5–10 min due to the rapid development of cracks, and transferred to the fragment soaking drops for 5–10 min from where they were fished and frozen.

ArgD crystals were soaked with fragments at a concentration of 50 mM overnight in otherwise the same manner as ArgC crystals grown in pH 5.5. A cryogenic solution was prepared by adding 30% ethylene glycol to the mother liquor. Crystals were briefly transferred to this solution and flash-frozen in liquid N₂.

ArgF crystals were soaked in drops containing crystallisation condition and 20 mM of ligand and equilibrated against 500 µL of reservoir solution for 16 h. A cryogenic solution was prepared by adding 25% ethylene glycol to the mother liquor. Crystals were briefly transferred to this solution and flash-frozen in liquid N₂.