Abstract
The bacterial flagellum is a large assembly of about 30 different proteins and is divided into three parts: the filament that acts as a screw propeller, the hook as a universal joint, and the basal body as a rotary motor. In the case of Salmonella, the filament length is 10–15 μm, which is more than ten times longer than the size of the cell. The filament is composed of only one component protein, flagellin, and is made of 11 protofilaments. The filament can form 12 different supercoiled structures as polymorphic forms. Each protofilament can take either the L (left-handed) or R (right-handed) state, and the number ratio of the protofilaments in these two states determines the shape of the supercoil. Some point mutations in flagellin make the filament straight by making all the protofilaments in one of the two states. The straight filaments enable us to use their helical symmetries for structural analysis by electron cryomicroscopy (cryoEM) and single particle image analysis. Here, we describe the methods for the purification of the flagellar filament and cryoEM data collection and image analysis.
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Acknowledgments
This work has been supported by JSPS KAKENHI Grant Number JP25000013 (to K.N.) and JP18K06155 (to T.M.). This work has also been supported by Platform Project for Supporting Drug Discovery and Life Science Research (BINDS) from AMED under Grant Number JP19am0101117 to K.N., by the Cyclic Innovation for Clinical Empowerment (CiCLE) from AMED under Grant Number JP17pc0101020 to K.N. and by JEOL YOKOGUSHI Research Alliance Laboratories of Osaka University to K.N.
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Yamaguchi, T., Miyata, T., Makino, F., Namba, K. (2023). Purification and CryoEM Image Analysis of the Bacterial Flagellar Filament. In: Minamino, T., Miyata, M., Namba, K. (eds) Bacterial and Archaeal Motility. Methods in Molecular Biology, vol 2646. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3060-0_5
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DOI: https://doi.org/10.1007/978-1-0716-3060-0_5
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