Chapter 12
In Situ Structural Analysis of Leptospira spp. by Electron
Cryotomography
Akihiro Kawamoto
Abstract
Spirochetes such as Treponema, Borrelia, and Leptospira species can rotate their bodies to swim in liquid
environments by rotating periplasmic flagella or endoflagella, which are present inside the cell. Electron
cryotomography (ECT) is an imaging technique that directly provides three-dimensional (3D) structures of
cells and molecular complexes in their cellular environment at nanometer resolution. Here, I present a
general protocol of ECT that covers the sample preparation, data collection, tilt series alignment, and
tomographic reconstruction for visualization of intact periplasmic flagella in Leptospira spp. This protocol is
capable of determining protein structures at resolutions high enough to visualize their individual domains
and secondary structures in their cellular environment.
Key words Electron cryotomography (ECT), Leptospira, Bacterial flagellum
1 Introduction
Bacteria such as Escherichia coli and Salmonella actively swim in
liquid environments by rotating long, helical, filamentous organelle
called the bacterial flagellum [1]. At the base of each filament, there
is a rotary motor powered by proton or other cation motive force
across the cytoplasmic membrane. The bacterial flagellum is a large
molecular assembly composed of nearly 30 different proteins that
identify it as an elaborate biological nanomachine [2]. While the
bacterial flagellum is generally present outside the cell body, the
spirochetes such as Treponema, Borrelia, and Leptospira species
have flagella in the periplasm of the cell [3–5]. The periplasmic
flagella generate swimming motility in liquid environments and
crawling motility on solid surfaces [6–8]. Interestingly, recent studies show that the periplasmic flagella determine the typical hookand spiral-shaped cell ends of Leptospira [9–11]. Structural analysis
of the periplasmic flagella is required for understanding their motility and cell shape determination mechanisms, but it is difficult to
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_12, © Springer Science+Business Media, LLC, part of Springer Nature 2020
131
In Situ Structural Analysis of Leptospira spp. by Electron
Cryotomography
Akihiro Kawamoto
Abstract
Spirochetes such as Treponema, Borrelia, and Leptospira species can rotate their bodies to swim in liquid
environments by rotating periplasmic flagella or endoflagella, which are present inside the cell. Electron
cryotomography (ECT) is an imaging technique that directly provides three-dimensional (3D) structures of
cells and molecular complexes in their cellular environment at nanometer resolution. Here, I present a
general protocol of ECT that covers the sample preparation, data collection, tilt series alignment, and
tomographic reconstruction for visualization of intact periplasmic flagella in Leptospira spp. This protocol is
capable of determining protein structures at resolutions high enough to visualize their individual domains
and secondary structures in their cellular environment.
Key words Electron cryotomography (ECT), Leptospira, Bacterial flagellum
1 Introduction
Bacteria such as Escherichia coli and Salmonella actively swim in
liquid environments by rotating long, helical, filamentous organelle
called the bacterial flagellum [1]. At the base of each filament, there
is a rotary motor powered by proton or other cation motive force
across the cytoplasmic membrane. The bacterial flagellum is a large
molecular assembly composed of nearly 30 different proteins that
identify it as an elaborate biological nanomachine [2]. While the
bacterial flagellum is generally present outside the cell body, the
spirochetes such as Treponema, Borrelia, and Leptospira species
have flagella in the periplasm of the cell [3–5]. The periplasmic
flagella generate swimming motility in liquid environments and
crawling motility on solid surfaces [6–8]. Interestingly, recent studies show that the periplasmic flagella determine the typical hookand spiral-shaped cell ends of Leptospira [9–11]. Structural analysis
of the periplasmic flagella is required for understanding their motility and cell shape determination mechanisms, but it is difficult to
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_12, © Springer Science+Business Media, LLC, part of Springer Nature 2020
131