The His-tag as a decoy modulating preferred orientation in cryoEM

Abstract

The His-tag is a widely used affinity tag that facilitates purification by means of affinity chromatography of recombinant proteins for functional and structural studies. We show here that His-tag presence affects how coproheme decarboxylase interacts with the water-air interface during grid preparation for cryoEM. Depending on His-tag presence or absence, we observe significant changes in patterns of preferred orientation. The analysis of particle orientations suggests that His-tag presence can mask the hydrophobic patches on a proteins surface that mediate the interactions with the water-air interface, while the hydrophobic linker between a His-tag and the coding sequence of the protein may enhance other interactions with water-air interface. Our observations suggest that tagging, including rational design of the linkers between an affinity tag and a protein of interest, offer a promising approach to modulating interactions with the water-air interface. SynopsisA His-tag affects the interactions of particles with the water-air interface in cryo-electron microscopy (cryoEM) single particle reconstruction (SPR), and thus may be used to modulate these interactions, including inducing changes in patterns of preferred orientation.