Crystallization of HheG wild type had previously been demonstrated using 10% (w/v) PEG4000 as precipitant, HEPES buffer with a pH of 7.3 to 7.5 and protein concentrations between 6 and 32 mg/ml. Hence, similar conditions were used to investigate the crystallizability of variants D114C and A221C (see supplementary for more details). This revealed that variant D114C displayed a higher crystallizability and required less PEG4000 compared to HheG wild type, while no crystals could be obtained for variant A221C (Table ). Moreover, variant D114C still gave the same hexagonal-shaped crystals as wild-type HheG (Figure ), but crystallized faster (Figure ) and formed overall larger crystals (Figure ). As the space group and cell parameters of D114C crystals were found to be the same or very similar to that of wild-type HheG (see below), the mutation at position 114 is likely responsible for improved crystallizability of variant D114C. In wild-type HheG, always two D114 residues of opposing tetramers face each other in the axial crystal interface, most likely resulting in a slight repulsion of the charged carboxyl side chains during crystallization. In contrast, the thiols in variant D114C (cysteine pKa = 8.1) are only partially charged at the pH used for crystallization.