a, Y2H assay with cell growth on agar plates containing 100 mM 3-AT, lacking tryptophan, leucine and histidine. Plates were imaged on day 5. Yellow, no growth on agar plates; light blue, weak growth forming non-circular colonies; dark blue, strong growth. b, Y2H result by growing yeast culture in liquid medium containing 100 mM 3-AT, lacking tryptophan, leucine and histidine. OD600 values were measured on day 2 to evaluate cell growth. c, An additional set of DHDs tested by Y2H showing improved orthogonality. d, Distribution of OD600 values for non-cognate interactions in b. The majority of cells grew to OD600 < 0.4, indicating weak interactions for non-cognate binding. e–g, Box plots of various properties for designs that assembled to off-target oligomeric states by native MS (failure) and that assembled into constitutive heterodimers (success). n = 88; 25th, 50th and 75th percentiles are shown in the box with the centre being median, extended to 1.5 × interquartile range (IQR) beyond the box. e, The number of buried bulky polar residues correlates strongly with design success. f, Successful designs tend to have a bigger polar interface surface area. g, Designs with better hydrophobic packing (as reported by the Rosetta filter value Average Degree on Ile, Leu and Val residues) tend to have a higher chance of being constitutive heterodimers as assessed by native MS. h, Contribution of bulky residues and hydrogen-bond networks to specific dimer formation. dSASA_polar measures interface hydrophilicity and correlates positively with the surface area of hydrogen-bond networks at the interface. Bulky polar residues in core counts the total number of buried bulky residues that participate in hydrogen-bond networks. Constitutive heterodimer formation (blue circles) or off-target oligomer formation (red circles) were determined with native MS. Filter cutoff values of dSASA_polar > 970 Å2 and more than one polar bulky residue buried in the core includes most of the successful designs and excludes most of the design failures. i, On the basis of the Y2H data in b, all 32 monomers from the 16 pairs were categorized as being specific (blue, has ≤1 non-cognate binding), or non-specific (red, has >1 non-cognate binding). With application of secondary structure prediction scores (PsiPred54) and Rosetta centroid energy score per residue as filters, designs with higher PsiPred values and lower Rosetta centroid score per residue are more specific (green box). Two independent experiments were performed (a–c).