Extended Data Fig. 5: Analyzing DHT03 heterotrimer with Rosetta and using structure prediction to determine base heterotrimer assembly.

a Computed energy landscape for DHT03, showing 10 replicate relaxes for the crystal structure (purple dots) and the original Rosetta design model (red dots). The RMSD to the un-relaxed crystal structure is computed across all Cα and plotted against Rosetta energy in Rosetta Energy Units (REUs). The relaxed design models have higher energies than the relaxed crystal structures. b The best-scoring relaxed crystal structure is compared to the best-scoring relaxed design model, showing energy differences for different score terms in Rosetta’s energy function. Negative values indicate that the score term favors the relaxed crystal structure. c Sidechain_rotamer and LJ terms are plotted on a per-residue level, with negative energy differences indicating that preference for the relaxed crystal structure arises from a summation of many small effects. d DHT03 predicted LDDT per residue is plotted for all 5 AlphaFold-Multimer³² models; Predicted Aligned Error (PAE) heat map for model 5 indicates low predicted Å error between pairs of residues across the interface. e Model 5 prediction (colored by pLDDT using a red-blue color spectrum) is aligned to DHT03 crystal structure (left) and the Rosetta design model (right), with a slightly better agreement to the design. f Single sequences for heterotrimer bases presented were used as input for AlphaFold-Multimer with the number of recycles set to 3. A representative prediction (colored by pLDDT using a red-blue color spectrum) with the lowest Cα RMSD is aligned to the design models colored by chain. Models predicting structures close to the designs are listed in parenthesis, with scores for AlphaFold confidence metrics pLDDT and pTM for all 5 AlphaFold-Multimer models reported in Supplementary Table 2 (the highest pLDDT and pTM scores indicate good agreement to DHT04, 05, 06, 08, 09, 10 design models). All 5 AlphaFold-Multimer models for DHT07 predict an inverted chain B.