Yi, G.; Mamalis, D.; Ye, M.; Carrique, L.; Fairhead, M.; Li, H.; Duerr, K. L.; Zhang, P.; Sauer, D. B.; von Delft, F.; Davis, B. G.; Gilbert, R. J. C.

Cryo-EM has become a routine structural biology method, yet elucidation of small proteins (<100 kDa) and increasing throughput remain challenging. Here, we describe covalently-dimerized engineered nanobodies, Di-Gluebodies, as novel and modular imaging scaffolds to address the challenges. They can simultaneously display two copies of the same protein or two distinct proteins, providing sufficient rigidity required by cryo-EM. We validate this method with multiple soluble and membrane targets, demonstrating that this provides a flexible and scalable platform for expanded protein structure determination.