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bioRxiv · 10.1101/2020.09.22.309005

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

Bromberg, R.·Guo, Y.·Plymire, D. A.·Emde, T.·Puzio, M.·Borek, D.·Otwinowski, Z.
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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.

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Bromberg, R., Guo, Y., Plymire, D. A., Emde, T., Puzio, M., Borek, D., Otwinowski, Z.. 2020-09-23. The His-tag as a decoy modulating preferred orientation in cryoEM. https://doi.org/10.1101/2020.09.22.309005

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