To unambiguously confirm the covalent binding of carbamates to the catalytic serine, a potent time-dependent hBChE inhibitor 6 was selected for crystallisation. After some trial-and-error, the careful selection of the crystal soaking conditions successfully led to the crystal structure of the dimethylcarbamoylated hBChE together in complex with the leaving group 5 at a 2.14-Å resolution (PDB 8AI7, Figure , left). Continuous electron density is observed close to Ser198 and the carbamate moiety (modelled here as a dimethylformamide molecule) can be fitted with a Ser198-Oγ DMF-carbon distance of 1.4 Å, in accordance with the formation of a covalent bond. While there are some similarities in the binding modes of the compounds from the previous series i.e., the cation-π interaction of the central tertiary amine of the leaving group with Tyr332, and a hydrophobic region above Trp82 in the choline-binding pocket that is occupied by the cycloheptyl ringthe indole moiety is this time predominantly facing the solvent (Figure , right). The reason for this is probably the steric hindrance with the dimethylcarbamoylated Ser198, where the methyl substituents encroach into the acyl-binding pocket that was previously occupied by a heterocyclic ring. The carbamoyl oxygen is nicely situated within the oxyanion hole, forming all three available hydrogen bonds with the backbone amide hydrogens of Gly116, Gly117 and Ala199. A water molecule (water 188) is located below His438 (modelled as protonated) and only 3.1 Å away from the carbonyl carbon of the dimethylcarbamate moiety, which is appropriately positioned to affect the hydrolysis and regeneration of the catalytic residue. The cycloheptyl still occupies the hydrophobic region above Trp82 in the choline-binding pocket, while the indole was displaced from the acyl-binding pocket and is predominantly facing the solvent (grey circlessolvent exposure).