Extended Data Fig. 4: RFdiffusion can condition on fold information to generate specific, thermostable folds.

A) 6WVS is a previously-described de novo designed TIM barrel (left). A fine-tuned RFdiffusion model can condition on 1D and 2D inputs representing this protein fold, specifically secondary structure (middle, bottom) and block-adjacency information (middle, top) (see Supplementary Methods 4.3.2). RFdiffusion then generates proteins that closely recapitulate this course-grained fold information (right). B) Outputs are diverse with respect to each other. With this coarse-grained fold specification, in silico successful designs are much more diverse (as quantified by pairwise TM-scores) compared to diversity generated through simply sampling many sequences for the original PDB backbone (6WVS). C) NTF2 folds are useful scaffolds for de novo enzyme design⁵⁶, and can also be readily generated with fold-conditioning in RFdiffusion. Designs are diverse and closely recapitulated by AF2. D) In silico success rates are high with fold-conditioned diffusion. TIM barrels are generated with an AF2 in silico success rate of 42.5% (left bar, pink) with in silico success incorporating both AF2 metrics and a TM-score vs 6WVS > 0.5. NTF2 folds are generated with an AF2 in silico success rate of 54.1% (right bar, pink), with in silico success incorporating both AF2 metrics and a TM-score vs PDB: 1GY6 > 0.5. In silico success was further validated with ESMFold (blue bars), where a pLDDT > 80 was used as the confidence metric for success. Gray: RFdiffusion design, colors: AF2 prediction. E) 11 TIM barrel designs were purified alongside the 6WVS positive control. Ten of these express and elute predominantly as monomers (note that the designs are approximately 4kDa larger than 6WVS). F) Eight designs expressed sufficiently for analysis by circular dichroism. All designs are folded, with circular dichroism spectra consistent with the designed structure (middle), and similar to 6WVS. Designs were also all highly thermostable, with CD melt analyses demonstrating designs were folded even at 95 °C (right). Designs are shown in gray, with the AF2 predictions overlaid in colors (left). Note that data on 6WVS and TIM_barrel_6 are duplicated from Fig. 2g, reproduced here for clarity.