Extended Data Fig. 6. External potentials for generating pockets around substrate molecules.

A–D) Example in silico successful designs for enzyme classes 2–5 (ref. ⁵⁷, see also Fig. 4). Native enzyme (PDB: 1CWY, 1DE3, 1P1X, 1SNZ); catalytic site (teal); RFdiffusion output (gray: model, colors: AF2 prediction). Metrics (AF2 vs design backbone r.m.s.d., AF2 vs design motif backbone r.m.s.d., AF2 vs design motif full-atom r.m.s.d., AF2 pAE): EC2: 0.93 Å, 0.50 Å, 1.29 Å, 3.51; EC3: 0.92 Å, 0.60 Å, 1.07 Å, 4.59; EC4: 0.93 Å, 0.80 Å, 1.03 Å, 4.41; EC5: 0.78 Å, 0.44 Å, 1.14 Å, 3.32. E–H) Implicit modeling of a substrate while scaffolding a retroaldolase active site triad [TYR1051-LYS1083-TYR1180] from PDB: 5AN7. E) The potential used to implicitly model the substrate, which has both a repulsive and attractive field (see Supplementary Methods 4.4). F) Left: Kernel densities demonstrate that without using the external potential (pink), designs often fall into two failure modes: (1) no pocket, and (2) clashes with the substrate. Right: clashes (substrate < 3 Å of the backbone) & pockets (no clash and > 16 Cα within 3–8 Å of substrate) with and without the potential. Two-proportion z-test: n = 71/51 +/− potential; clashes z = −2.05, p = 0.02, pocket z = −2.27, p = 0.01. Each datapoint represents a design already passing the stringent in silico success metrics (AF2 motif r.m.s.d. < 1 Å, AF2 backbone r.m.s.d. < 2 Å, AF2 pAE < 5). Note that the potential and clash definition pertain only to backbone Cα atoms, and do not currently include sidechain atoms. G) Designs close to the labeled local maxima of the kernel density estimate. Without the potential, the catalytic triad is predominantly (1) exposed on the surface with no residues available to provide substrate stabilization or (2) buried in the protein core, preventing substrate access. With the potential, the catalytic triad is predominantly (3), partially buried in a concave pocket with shape complementary to the substrate. Backbone atoms within 3 Å of the substrate are shown in red. H) A variety of diverse designs with pockets made using the potential, with no clashes between the substrate and the AF2-predicted backbone. The functional form and parameters used for the pocket potential are detailed in Supplementary Methods 4.4. In each case the substrate is superimposed on the AF2 prediction of the catalytic triad.