Chapter 4
In Situ Imaging and Structure Determination
of Biomolecular Complexes Using Electron
Cryo-Tomography
Mohammed Kaplan, William J. Nicolas, Wei Zhao, Stephen D. Carter,
Lauren Ann Metskas, Georges Chreifi, Debnath Ghosal,
and Grant J. Jensen
Abstract
Electron cryo-tomography (cryo-ET) is a technique that allows the investigation of intact macromolecular
complexes while they are in their cellular milieu. Over the years, cryo-ET has had a huge impact on our
understanding of how large biomolecular complexes look like, how they assemble, disassemble, function,
and evolve(d). Recent hardware and software developments and combining cryo-ET with other techniques,
e.g., focused ion beam milling (FIB-milling) and cryo-light microscopy, has extended the realm of cryo-ET
to include transient molecular complexes embedded deep in thick samples (like eukaryotic cells) and
enhanced the resolution of structures obtained by cryo-ET. In this chapter, we will present an outline of
how to perform cryo-ET studies on a wide variety of biological samples including prokaryotic and
eukaryotic cells and biological plant tissues. This outline will include sample preparation, data collection,
and data processing as well as hybrid approaches like FIB-milling, cryosectioning, and cryo-correlated light
and electron microscopy (cryo-CLEM).
Key words Cryo-ET, In situ structural biology, Subtomogram averaging, FIB-milling, High-resolution tomography, Cryosectioning
1 Introduction
The continuous interaction of proteins with each other, and with
other molecules, in the crowded environment of the cell generates
life. Disentangling the structure and dynamics of proteins while
they are in this native milieu is crucial to structural biology. To
achieve this, multiple imaging and spectroscopic techniques have
been developed in recent years that aim at investigating biomolecules at ever higher resolution inside the cell (see, e.g., Refs. 1–4).
Cryo-ET has now become an indispensable tool to investigate
molecular complexes in intact cells in a frozen hydrated state at
Tamir Gonen and Brent L. Nannenga (eds.), CryoEM: Methods and Protocols, Methods in Molecular Biology, vol. 2215,
https://doi.org/10.1007/978-1-0716-0966-8_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021
83
In Situ Imaging and Structure Determination
of Biomolecular Complexes Using Electron
Cryo-Tomography
Mohammed Kaplan, William J. Nicolas, Wei Zhao, Stephen D. Carter,
Lauren Ann Metskas, Georges Chreifi, Debnath Ghosal,
and Grant J. Jensen
Abstract
Electron cryo-tomography (cryo-ET) is a technique that allows the investigation of intact macromolecular
complexes while they are in their cellular milieu. Over the years, cryo-ET has had a huge impact on our
understanding of how large biomolecular complexes look like, how they assemble, disassemble, function,
and evolve(d). Recent hardware and software developments and combining cryo-ET with other techniques,
e.g., focused ion beam milling (FIB-milling) and cryo-light microscopy, has extended the realm of cryo-ET
to include transient molecular complexes embedded deep in thick samples (like eukaryotic cells) and
enhanced the resolution of structures obtained by cryo-ET. In this chapter, we will present an outline of
how to perform cryo-ET studies on a wide variety of biological samples including prokaryotic and
eukaryotic cells and biological plant tissues. This outline will include sample preparation, data collection,
and data processing as well as hybrid approaches like FIB-milling, cryosectioning, and cryo-correlated light
and electron microscopy (cryo-CLEM).
Key words Cryo-ET, In situ structural biology, Subtomogram averaging, FIB-milling, High-resolution tomography, Cryosectioning
1 Introduction
The continuous interaction of proteins with each other, and with
other molecules, in the crowded environment of the cell generates
life. Disentangling the structure and dynamics of proteins while
they are in this native milieu is crucial to structural biology. To
achieve this, multiple imaging and spectroscopic techniques have
been developed in recent years that aim at investigating biomolecules at ever higher resolution inside the cell (see, e.g., Refs. 1–4).
Cryo-ET has now become an indispensable tool to investigate
molecular complexes in intact cells in a frozen hydrated state at
Tamir Gonen and Brent L. Nannenga (eds.), CryoEM: Methods and Protocols, Methods in Molecular Biology, vol. 2215,
https://doi.org/10.1007/978-1-0716-0966-8_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021
83