The risk of unexpected complexes and oxidations like this is not specific to the structures discussed here. The conditions that led to these reactions, in both this case and previously , are widely used. Many common methods for the cleavage of fusion proteins (thrombin, factor Xa, tobacco etch virus protease, and rhinovirus 3C protease) leave glycine as the N-terminal residue . Unsurprisingly then, the N-terminal Gly-Xaa-His motif is common in the Protein Data Bank, appearing in >7,000 sequences (~4% of the total). Nickel affinity columns are also widely used. Many of these proteins would therefore be expected to form Gly-Xaa-His-Ni 2+ complexes. However, the first few residues of the N-terminus are almost invariably disordered: 97% of human proteins have disordered terminal residues , and 42% of all disordered residues are in the N-terminus . Thus, these complexes are very unlikely to be resolved, and are therefore likely to go undetected. Peroxide-generating buffers such as HEPES are also ubiquitous; thus, quite common procedures for protein preparation inadvertently generate ROS. Oxidation by ROS can have many undesirable effects on proteins, from modifying side chains (which may influence the overall conformation) to cleaving the amide backbone .