MODULAR PROTEIN-DNA CO-CRYSTALS FOR PROGRAMMABLE SCAFFOLD ASSISTED STRUCTURE DETERMINATION

Abstract

The structure of biomolecules can provide key insight into understanding their underlying function. X-ray crystallography is a principal technique in structural biology used to determine atomic-level details of macromolecular structures. Despite major advances in X-ray diffraction instrumentation and computational modelling, the practical bottleneck in crystallography continues to be crystallization itself. Scaffold assisted crystallography offers an alternative to approach to traditional crystallization. Instead of crystallizing each target independently, one could grow a robust crystalline framework in advance and subsequently introduce guest molecules for structure determination. To be practically useful, such scaffold crystals must be highly porous, modular, and mechanically stable under diverse solvent conditions. Here, a family of co-crystals (CC1) was systematically expanded through lattice engineering of its DNA and protein constituents. The programmable nature of DNA enabled rational modulation of lattice dimensions, pore architecture, and the incorporation of tailored sequence motifs that serve as anchoring sites for guest DNA-binding proteins. At the same time, the protein component was engineered through point mutations and sequence fusions—changes that could disrupt crystal formation—yet the lattice retained its overall topology and packing geometry. This combined DNA- and protein-level modularity provides an unusually broad design landscape for a crystalline system, establishing CC1 as a robust and adaptable scaffold for subsequent functional applications. Finally, CC1 scaffold crystal variants were applied to the long-standing challenge of post-crystallization guest structure determination. Stabilized crystals accommodated substantial changes in solvent environment and enabled a range of DNA-binding proteins to diffuse into the lattice and bind their cognate sites in an ordered fashion. These bound guests produced clear electron density suitable for structural analysis. This achievement represents the first successful use of a porous protein–DNA co-crystal to determine the structure of guest macromolecular added via simple soaking, providing a definitive demonstration of scaffold-assisted crystallography and establishing CC1 as a foundational platform for future structural biology and biomaterials applications.