X‐ray crystal structure of a designed rigidified imaging scaffold in the ligand‐free conformation

Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by cryogenic electron microscopy (cryo‐EM). One particularly well characterized scaffold type is a symmetric tetrahedral assembly composed of 24 subunits, 12 A and 12 B, which has three cargo‐binding DARPins positioned on each vertex. Here, the X‐ray crystal structure of a representative tetrahedral scaffold in the apo state is reported at 3.8 Å resolution. The X‐ray crystal structure complements recent cryo‐EM findings on a closely related scaffold, while also suggesting potential utility for crystallographic investigations. As observed in this crystal structure, one of the three DARPins, which serve as modular adaptors for binding diverse `cargo' proteins, present on each of the vertices is oriented towards a large solvent channel. The crystal lattice is unusually porous, suggesting that it may be possible to soak crystals of the scaffold with small (≤30 kDa) protein cargo ligands and subsequently determine cage–cargo structures via X‐ray crystallography. The results suggest the possibility that cryo‐EM scaffolds may be repurposed for structure determination by X‐ray crystallography, thus extending the utility of electron‐microscopy scaffold designs for alternative structural biology applications. The structure of an imaging scaffold engineered to bind and study therapeutic protein targets has been determined at 3.8 Å resolution. Cargo protein‐binding DARPins are positioned within the large solvent channels of an unusually porous crystal lattice, suggesting that it may be possible to soak crystals with small target proteins in order to determine their structures.