Improved cryo-EM reconstruction of sub-50 kDa complexes using 2D template matching

Visualizing the structures of small proteins and complexes has been a longstanding challenge in single-particle cryo-EM. Some of these targets have been successfully resolved by binding to antibody fragments (Fabs) or fusing with external scaffolds to increase their size. Recent advances in conventional single-particle techniques have enabled the determination of an in-creasing number of structures smaller than 100 kDa, achieving resolutions relevant to drug research. Compared to X-ray crystallography, cryo-EM preserves the near-native states of biomolecules, can resolve structural heterogeneity, and has the potential to apply to a wide range of targets. In this work, we demonstrate that the alignment and reconstruction of small macromolecular complexes can be significantly improved using high-resolution structures as priors combined with 2D template matching. Using this method, we were able to reconstruct previously intractable targets under 50 kDa and improve the density of the ligand-binding sites in the reconstructions. We envision that this method will further lower the molecular weight limit of single-particle cryo-EM, enabling its application to important drug-binding complexes that are below 50 kDa.