As an example for demonstrating evaluation with MotifBench and to stand as a baseline for evaluation of novel motif-scaffolding methods we have designed scaffolds each benchmark problem with RosettaFold Diffusion and uploaded these scaffolds to Zenodo at zenodo.org/records/14731790.

The below provides details and scripts about how this scaffold set was generated.

1. Choosing "contigs" in contig_specifications.csv

RFdiffusion requires the specification of a "contig" that describes the placement of the sequence-contiguous motif segments in the scaffold to be generated. This choice of contig can have significant implications on the scaffolds that it generates. For motifs problems that were part of the earlier RFdiffusion benchmark set, we adopt the contig used there.

For the problems with one segment we chose the contig to place no limits on the size of the flanking regions.

For the remaining problems we chose contigs that specified order of segments in the reference structure and included gaps between segments of at least 10 residues that spanned the gap in the reference structure between the associated segments.

2. Running RFdiffusion motif-scaffolding via apptainer and docker image

• We installed RFdiffusion using the docker image (see readme, accessed November 2024).
• We used generated SLURM scripts for each benchmark problem using write_scripts_by_case.sh. The script includes details of the exact RFdiffusion run commands.
• We ran these on a university compute cluster, Sherlock. Each script ran on a single GPU, of which the model varied. The mean runtime across test cases to generate 100 scaffolds was 62 minutes. Per-problem runtimes are reported in runtime_by_problem.txt.

3. Assembly of scaffold_info.csv for each benchmark problem.

Finally we generated the scaffold_info.csv metadata files for each problem with write_all_scaffold_info_csvs.sh.

Reference PDB evaluation results

We additionally provide intermediate computations and results of the evaluation of reference scaffolds from which motifs are defined in reference_pdb_baseline/.