As the previous cross-linking of HheG crystals with the commonly used cross-linker glutaraldehyde significantly reduced enzyme activity, protein engineering of HheG was used herein to introduce desired amino acids on the enzyme surface as new cross-linking sites for alternative cross-linkers. Specifically, we aimed for the insertion of cysteine residues, as HheG wild type contains only few cysteines that could potentially be cross-linked as well. Thus, the available crystal structure of wildtype HheG was analyzed for potential residues facing each other in the crystal interface. As shown in Figure , two major interfaces (contact points) between HheG tetramers within the crystal can be distinguished: an axial and a tangential interface. The axial interface refers to crystal contacts between tetramers lining up in infinite strings throughout the crystal, while the tangtential interface represents crystal contacts between tetramers of orthogonal strings. Existing crystal contacts were analyzed using the PRODIGY crystal webserver to identify residues with less than 5 Å distance to each other. Interestingly, this revealed three residues with the same amino acid positions of different HheG tetramers facing each other in the crystal interfaces: M45 and D114 in the axial interface, and A221 in the tangential interface (Figure ). Those residues were selected for replacement by cysteine, as mutagenesis of one residue would directly result in the required cysteine pairs of neighbouring tetramers for later cross-linking. As the Cα distances of those opposing residues are between 6.5-8.5 Å (Table ), bis-maleimidoethane (BMOE) with a spacer length of 8 Å was selected as thiol-specific crosslinker for later CLEC generation (Figure ). HheG variants M45C, D114C and A221C were generated, produced in E. coli and purified according to established protocols for wild-type HheG. Corresponding yields after purification are listed in Table .