Extended Data Fig. 2: Analysis of the critical steps of the de novo binder design pipeline.

a, Comparison of the two docking approaches based on Rosetta ddG and contact molecular surface. Average and per-target distribution of the top 1% of binders in two key metrics after pooling equal-CPU-time dock-and-design trajectories. RifDock seeded with PatchDock outputs generated 300 outputs per scaffold that were trimmed to a total of 19,500 docks with “The Predictor” and designed using combinatorial side-chain optimization (orange). RifDock using the Hierarchical docking search generated 300 outputs per scaffold that were trimmed to a total of 19,500 docks with “The Predictor” and subsequently designed (purple). Rosetta ddG refers to the predicted binding energy as calculated by Rosetta and Contact MS to key residues refers to the Contact Molecular Surface value (a distance weighted interfacial area calculation) to the key hydrophobic residues on the target that define this binding site. b, The rapid pre-screening method enriches docks with better Rosetta ddG and contact molecular surface. Average and per-target distribution of the top 1% of binders in two key metrics after pooling equal-CPU-time dock-and-design trajectories. The top 30 PatchDock outputs for the 1,000 helical scaffolds tested were designed using the RosettaScripts protocol (blue). The top 300 PatchDock outputs for the 1,000 helical scaffolds tested were trimmed to 21,000 with “The Predictor” and subsequently designed (red). c, The improved sequence design protocol yielded amino acid sequences more strongly predicted to fold to the monomer structure. The effect on fragment quality and Rosetta Score with different fragment-quality-guidance approaches. Rosetta using FastDesign with the standard LayerDesign settings was used to design 1,000 3-helical and 1,000 4-helical mini-protein scaffolds (blue). The same protocol was supplanted with the ConsensusLoopDesign TaskOperation (orange). The structure-based PSSM was used as an energy term in addition to the Standard Rosetta protocol (green). Two predictors of sequence-structure correspondence were found to improve without negatively affecting the computed Rosetta score of the binders. The probability that the designed sequence encoded for the wrong secondary structure was computed using PsiPred4⁸⁸ (left), and for each 9aa fragment of the designed scaffold, the closest match to a fragment in the Protein Data Bank with the same sequence was computed and averaged over the entire structure¹⁰ (center). Details can be found in the Supplemental Information. d, The improved sequence design protocol yielded amino acid sequences more strongly bound to the target. 10,000 scaffolds docked against the N-terminal domain of EGFR were designed with the RosettaScripts protocol while varying only the weight of the ProteinProteinInterfaceUpweighter. This TaskOperation multiplies all energies across the interface by the listed value during packing-design calculations.