Gluebodies improve crystal reliability and diversity through transferable nanobody mutations that introduce constitutive crystal contacts

Obtaining protein crystals suitable for characterizing ligands and understanding different protein conformations remains a bottleneck for structural studies. Using nanobodies as crystallization chaperones is one strategy to address the problem, but its reliability is uncharacterized and in this study, we observed it to have a poor success rate. We therefore set out to engineer robust crystallization behaviour into the nanobody scaffold by exploring the nanobody-nanobody interface predominant in the PDB using >200 combinations of surface mutations. This yielded multiple polymorphs, all mediated by this interface, with one providing far superior resolution and reliability of diffraction. We therefore refer to the modified nanobody scaffolds as “Gluebodies” and propose at least 2 variants that should be routinely generated for chaperone experiments. We furthermore show that Gluedodies cannot rescue intrinsically non-crystallizing proteins, but are a powerful approach to improve the packing and resolution limit of poorly diffracting crystals.