De novo design of semisynthetic conduction pores

Key Points:

• Researchers used sequential MASTER searches on a non-redundant PDB30 dataset to design H2 backbone models, selecting three based on structural prevalence and interface geometry for further loop building.
• The helix-loop-helix motif connecting the Anchor and H2 helices was designed via MASTER loop searches with varying residue gaps, selecting models with the lowest RMSD for designability analysis.
• Single-channel electrical recordings of various (CsgG:CsgX)9 complexes demonstrated distinct pore current profiles, with Gaussian fits indicating differences among constructs.
• Cryo-EM structural validation of multiple (CsgG:CsgX)9 variants revealed detailed pore architectures, local resolution maps, and density supporting the presence of designed motifs, though some variants lacked density beyond certain residues.
• Attempts to design CsgX-like extensions using RFdiffusion were unsuccessful, producing either inward-folding elongations or further Anchor elongations, leading researchers to favor a data-driven design approach based on PDB structural information for optimal interface construction.