3.2.3. Two-Layer Approach. The need for four-body terms drastically increases the requisite number of subsystem calculations. Automatic energy screening using GFN2-xTB, which was developed in parallel with this work, should ultimately be useful in this regard. As an alternative, we examine the use of a two-layer supersystem correction (eq. 7). For the calculation of thermochemical quantities in enzymes, this approach worked quite well when used with only a two-body expansion at the higher level of theory, and low-level methods such as HF/6-31G. Versions of this "MIM2" procedure (and also a three-layer "MIM3") have been used in other fragment-based calculations of protein-ligand interaction energies, typically using the semi-empirical PM6+D3 144,145 method for the low-level correction, and with smaller (sub-residue) fragments as compared to the present work. Here, we examine the efficacy of using HF-3c to compute δ frag in eq. 7, since we know that HF-3c is computationally feasible in very large enzyme models.