Chapter 13
MicroED Sample Preparation and Data Collection For
Protein Crystals
Guanhong Bu and Brent L. Nannenga
Abstract
Microcrystal Electron Diffraction (MicroED) enables structure determination of very small crystals that are
much too small to be of use for other conventional diffraction techniques. MicroED has been used to
determine the structures of many proteins and small organic molecules, and the technique can be
performed on most standard cryo-TEM instruments equipped with high-speed detectors capable of
collecting electron diffraction data. Here, we present protocols for MicroED sample preparation and data
collection for protein microcrystals.
Key words Microcrystal electron diffraction, MicroED, Protein crystallography, Structural biology,
Sample preparation
1 Introduction
Microcrystal electron diffraction (MicroED) is a method for collecting electron diffraction data using a cryo-transmission electron
microscope (cryo-TEM) [1, 2]. MicroED makes it possible to
collect high-resolution electron diffraction data sets from extremely
small three-dimensional crystals that would otherwise be unusable
for conventional X-ray diffraction methods [3, 4]. Since its initial
development, MicroED has been successfully used on a variety of
protein [5–10], peptide [11–16], and small molecule targets [17–
21]. Sample preparation methods can vary depending on the type
of crystal being studied, and here we will present methods for
preparing protein crystals samples for MicroED analysis.
When working with protein samples, it is critical that the crystals remain hydrated; therefore, protein microcrystals are vitrified in
liquid ethane in order to ensure they are preserved in a frozen
hydrated state within the cryo-TEM. Sample preparation equipment and procedures for MicroED are similar to those used in
single
particle
cryo-electron
microscopy
(cryo-EM)
[22, 23]. Briefly, microcrystals are applied onto a carbon-coated
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_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021
287
MicroED Sample Preparation and Data Collection For
Protein Crystals
Guanhong Bu and Brent L. Nannenga
Abstract
Microcrystal Electron Diffraction (MicroED) enables structure determination of very small crystals that are
much too small to be of use for other conventional diffraction techniques. MicroED has been used to
determine the structures of many proteins and small organic molecules, and the technique can be
performed on most standard cryo-TEM instruments equipped with high-speed detectors capable of
collecting electron diffraction data. Here, we present protocols for MicroED sample preparation and data
collection for protein microcrystals.
Key words Microcrystal electron diffraction, MicroED, Protein crystallography, Structural biology,
Sample preparation
1 Introduction
Microcrystal electron diffraction (MicroED) is a method for collecting electron diffraction data using a cryo-transmission electron
microscope (cryo-TEM) [1, 2]. MicroED makes it possible to
collect high-resolution electron diffraction data sets from extremely
small three-dimensional crystals that would otherwise be unusable
for conventional X-ray diffraction methods [3, 4]. Since its initial
development, MicroED has been successfully used on a variety of
protein [5–10], peptide [11–16], and small molecule targets [17–
21]. Sample preparation methods can vary depending on the type
of crystal being studied, and here we will present methods for
preparing protein crystals samples for MicroED analysis.
When working with protein samples, it is critical that the crystals remain hydrated; therefore, protein microcrystals are vitrified in
liquid ethane in order to ensure they are preserved in a frozen
hydrated state within the cryo-TEM. Sample preparation equipment and procedures for MicroED are similar to those used in
single
particle
cryo-electron
microscopy
(cryo-EM)
[22, 23]. Briefly, microcrystals are applied onto a carbon-coated
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_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021
287