To determine crystallization parameters, crystallization was scaled up to 20 µl. Now, sitting-drop crystallization was performed in 20 µl droplets containing optimized protein solution mixed with crystallization buffer (1:1) (Table ) on cover glass plates. For wild-type HheG, a protein concentration of 32 mg/ml was used in contrast to 24 mg/ml for variant HheG D114C. As reservoir, 3 ml crystallization buffer were placed in a separate, smaller petri dish. For 72 h at 8 °C, droplets (on cover glass) and reservoir (in small petri dish) were placed in a standard petri dish that was sealed with parafilm until microscopic documentation.