A novel "bio-tag" for cryo-EM studies based on the small, electron-dense protein Csp1

Small proteins can be challenging to study by single-particle cryogenic electron microscopy (cryo-EM) techniques because they have low signal-to-noise ratios, making them difficult to identify and analyze. Here we investigated the use of Csp1, a small (∼50 kDa) tetrameric metal-binding protein, to act as a "bio-tag" to help overcome this problem. We find Csp1 is compact, stable, and exhibits enhanced electron scattering and excellent particle contrast in cryo-EM micrographs. As a result, we could determine the structure of Csp1 to 2.98-Å resolution using standard cryo-EM approaches. We also tested if Csp1 could be used as a tag or fiducial to help determine the structure of a protein bound to it. Specifically, we analyzed an epitope-tagged Csp1 bound to a ∼40 kDa Fab fragment from the antibody 1D4. Data from these complexes yielded medium-resolution structures of the complex (5.70 Å) and the bound 1D4 Fab (5.40 Å). These results suggest that, with further optimization, electron-rich Csp1 is a promising bio-tag for use in cryo-EM studies.