We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. Here we use trimeric protein building blocks to design a 24-subunit, 13 nm diameter complex with octahedral symmetry and two related variants of a 12-subunit, 11 nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.

What conformations do protein molecules populate in solution? Crystallography provides a highresolution description of protein structure in the crystal environment, while NMR describes structure in solution but using less data. NMR structures display more variability, but is this because crystal contacts are absent or because of fewer data constraints? Here we report unexpected insight into this issue obtained through analysis of detailed protein energy landscapes generated by large-scale, native-enhanced sampling of conformational space with Rosetta@HOME for 111 protein domains. In the absence of tightly associating binding partners or ligands, the lowest-energy Rosetta models were nearly all <2.5Å C α RMSD from the experimental structure; this result demonstrates that structure prediction accuracy for globular proteins is limited mainly by the ability to sample close to the native structure. While the lowest-energy models are similar to deposited structures, they are not identical; the largest deviations are most often in regions involved in ligand, quaternary, or crystal contacts. For ligand binding proteins, the low energy models may resemble the apo structures, and for oligomeric proteins, the monomeric assembly intermediates. The deviations between the low energy models and crystal structures largely disappear when landscapes are computed in the context of the crystal lattice or multimer. The computed low-energy ensembles, with tight crystal-structure-like packing in the core, but more NMR-structure-like variability in loops, may in some cases resemble the native state ensembles of proteins better than individual crystal or NMR structures, and can suggest experimentally testable hypotheses relating alternative states and structural heterogeneity to function.

We describe RosettaRemodel, a generalized framework for flexible protein design that provides a versatile and convenient interface to the Rosetta modeling suite. RosettaRemodel employs a unified interface, called a blueprint, which allows detailed control over many aspects of flexible backbone protein design calculations. RosettaRemodel allows the construction and elaboration of customized protocols for a wide range of design problems ranging from loop insertion and deletion, disulfide engineering, domain assembly, loop remodeling, motif grafting, symmetrical units, to de novo structure modeling.

The type III secretion system (T3SS) is a macromolecular ‘injectisome’, that allows bacterial pathogens to transport virulence proteins into the eukaryotic host cell. This macromolecular complex is constituted by connected ring-like structures that span both bacterial membranes. The crystal structures of the periplasmic domain of the outer membrane (OM) secretin EscC and the inner membrane (IM) protein PrgH reveal the conservation of a modular fold among the three proteins which form the OM and IM rings of the T3SS. This leads to the hypothesis that this conserved fold provides a common ring-building motif that allows for the assembly of the variably sized OM and IM rings characteristic of the T3SS. Utilizing an integrated structural and experimental approach, ring-models for the periplasmic domain of EscC were generated and placed in the context of the assembled T3SS, providing evidence for direct interaction between the OM and IM ring components and an unprecedented span of the OM secretin.