Fig. 1: Overview of the design approach.

a, Scaffold sampling for base heterotrimer design. (i) To generate ABC coiled coils, the three helical parameters (radius (R; double-headed arrows), rotation around the helical axis (Δɸ; curved arrows) and relative displacement along the z axis (Z_off; square brackets)) are independently sampled for each of the three helices (eight parameters in total, as the Z offset for the first helix is zero). (ii) These backbones are then coupled to Monte Carlo HBNet to find hydrogen bond networks spanning all three helices. For the helical bundle approach, to break down the combinatorial explosion that arises when all 6 × 3 − 1 = 17 helical parameters are sampled simultaneously, a stepwise approach is taken to divide the search problem into three steps. (1) Backbone sampling is carried out for the three inner helices and one outer helix and Monte Carlo HBNet is used to identify the subset of backbones that can host a network spanning all four helices. (2) For this subset, a fifth helix is then sampled and Monte Carlo HBNet is used to identify backbones with networks that span this helix and the three central helices. (3) To these selected backbones, a sixth helix is added and a final Monte Carlo HBNet search is carried out to identify networks involving this new helix and the three inner helices. (iii) Backbones from both approaches can be optionally trimmed, packed with phenylalanine and other aliphatic residues in the core and decorated with charged residues at the surface to enhance electrostatic interaction across the chains. (iv) To the six-helix bundles, short designed connecting loops are added to generate three helical hairpin units. Two possible loop combinations are shown, with clockwise or counterclockwise closure and with loops all facing the same direction (solid lines) or loops at opposing terminal ends (dashed lines). b, Designed helical repeat (DHR) monomers can be rigidly joined to both coiled coil and helical bundle heterotrimers through single fusions, which can then be combined to make four- and three-arm heterotrimers, respectively. c, Heterotrimer arms can be combined with other designed building blocks to create higher-order nanostructures, such as A2B2 heterotetramers or A3B3C3/A4B4C4 hetero-oligomers.