When the fragment calculations required for MBE(n) are performed using PCM boundary conditions, and results compared to a supramolecular calculation with the same boundary conditions, we recover good convergence of MBE(n) even for single-residue fragments having net charge. Results for several other DFT functionals and basis sets are provided in Tables and, and in Fig. we extend some of these results to n = 4 in order to check convergence. Using PCM boundaries, the difference between the MBE(3) and MBE(4) results is 1 kcal/mol, while gas-phase calculations sometimes afford errors > 150 kcal/mol at the four-body level! MBE(3) calculations with low-dielectric boundary conditions consistently provide sub-kcal/mol accuracy for various functionals and basis sets, whereas MBE(3) with vacuum boundary conditions affords errors of 10-30 kcal/mol in many cases. Notably, we obtain stable results even when the basis set contains diffuse functions. These can be problematic when self-consistent charge-embedding schemes are used. These results suggest that large errors for enzymatic thermochemistry obtained using MBE(n) with vacuum boundary conditions originate not from the fragmentation approximation itself, or from the simple hydrogen atom caps that we use to saturate the severed valencies upon fragmentation. (This is less sophisticated as compared to "conjugated caps" that try to replicate amino acid moieties, Figure : Multilayer techniques applied to the complex of an enzyme (depicted as a chain of amino acids) and a substrate (labeled "S"). Colors encode the level of theory, with the higher-level method in orange and the lower-level method in blue. (a) Conventional ONIOM method, in which high-level calculations are applied only to the model system (b) Multilayer fragmentation method, where the high-level method is applied to the entire system by means of fragmentation.