Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.06.325969v1?rss=1 Authors: Tamura-Sakaguchi, R., Aruga, R., Hirose, M., Ekimoto, T., Miyake, T., Hizukuri, Y., Oi, R., Kaneko, M. K., Kato, Y., Akiyama, Y., Ikeguchi, M., Iwasaki, K., Nogi, T. Abstract: Antibody labeling has been extensively conducted for structure determination in both x-ray crystallography and EM analysis. However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, we have developed an alternative method to prepare an antibody-complex by inserting an exogenous epitope into the target. We here demonstrated that the Fab of monoclonal antibody NZ-1 could form a stable complex with the target containing an inserted PA14 tag as an epitope. By adopting a closed ring-like conformation inside the antigen-binding pocket, the inserted PA14 tag had less impact on the folding of the target. In fact, the PA14 tag could also be inserted into the sterically hindered loop for labeling. Using the improved labeling technique, we performed negative-stain EM on a bacterial site-2 protease, which enabled us to approximate the domain arrangement based on the docking mode of the NZ-1 Fab. Copy rights belong to original authors. Visit the link for more info