Significant advances in the past decade have enabled high-resolution structure determination of a vast variety of proteins by cryogenic electron microscopy single particle analysis. Despite improved sample preparation, next-generation imaging hardware, and advanced single particle analysis algorithms, small proteins remain elusive for reconstruction due to low signal-to-noise and lack of distinctive structural features. Multiple efforts have therefore been directed at the development of size-increase techniques for small proteins. Here we review the latest methods for increasing effective molecular weight of proteins <100 kDa through target protein binding or target protein fusion - specifically by using nanobody-based assemblies, fusion tags, and symmetric scaffolds. Finally, we summarize these state-of-the-art techniques into a decision-tree to facilitate the design of tailored future approaches, and thus for further exploration of ever-smaller proteins that make up the largest part of the human genome.
Keywords: BRIL, cytochromeb562 RIL; DARPin, Design Ankyrin Repeat Protein; Fab, antigen binding fragment; GFP, Green Fluorecent Protein; GPCR, G protein-coupled receptor; MW, molecular weight; Mb, megabody; Nb, nanobody; SNR, signal-to-noise ratio; SPA, single particle analysis; TM, transmembrane; cryo-EM, cryogenic electron microscopy; kDa, kiloDalton; κOR ICL3, κ-opiod receptor intracellular loop 3.
© 2022 The Authors.