(A) Ribbon diagram illustrating the structure of α-glu II with the key catalytic residues shown as sticks. The catalytic subunit is shown in gold, whilst the fragment of the accessory subunit remaining after protease digestion is shown in silver. (B) The catalytic binding site of α-glu II. Key residues for catalytic activity and interaction with the substrate glycan are shown as sticks. In the catalytic cycle D564 acts as a nucleophile and D640 as the catalytic acid/base. As is typical for sugar-binding sites, there are many aromatic residues and polar groups offering a wide range of potential interactions for inhibitors. (C) The catalytic binding site of α-glu II with NB-DNJ shown in magenta as a ball-and-stick representation. In this structure, the glucose-like portion occupies a similar position to the natural substrate and the alkyl chain occupies the +1 site, which would be occupied by the sugar moiety adjacent to that cleaved in the reaction in the natural substrate. The side chain of W525 becomes disordered and is not shown in the structure. (D) The catalytic binding site of α-glu II with the inhibitor MON-DNJ shown in purple as a ball-and-stick representation. In this structure, the alkyl chain of the iminosugar fully occupies the +1 site and extends beyond; the alkyl chain adopts two main conformations, one of which is located towards the +2 sugar-binding site. Addition of the inhibitor leads to disorder in the catalytic site including displacement of the 523–528 loop (containing W525) and multiple conformations for F571. In all figures, H atoms are removed for clarity and all structures are shown in the same orientation. Protein databank file 5foe.pdb was shown in A and B; 5ieg.pdb and 5ief.pdb were used for C and D, respectively.