Chapter 14
Detection of Microcrystals for CryoEM
Simon Weiss, Sandra Vergara, Guowu Lin, and Guillermo Calero
Abstract
Here, we present a strategy to identify microcrystals from initial protein crystallization screen experiments
and to optimize diffraction quality of those crystals using negative stain transmission electron microscopy
(TEM) as a guiding technique. The use of negative stain TEM allows visualization along the process and
thus enables optimization of crystal diffraction by monitoring the lattice quality of crystallization conditions. Nanocrystals bearing perfect lattices are seeded and can be used for MicroED as well as growing
larger crystals for X-ray and free electron laser (FEL) data collection.
Key words Protein crystallization, Optimization, Brightfield microscopy, UV microscopy, Negative
staining TEM, FFT-calculation, Granular aggregate, Microcrystal, Crystal lattice quality
1 Introduction
Crystallization of protein targets remains the most significant challenge in the process of structure determination by macromolecular
crystallography. When setting up crystallization trays experimenters
can obtain three different negative outcomes: (a) conditions with
amorphous precipitate, (b) few or no conditions with apparent
crystals or (c) crystals that do not diffract or diffract to low resolution. To tackle these problems most recent efforts have focused
mainly on improving sample “crystallizability” by implementing
techniques such as alanine scanning mutagenesis [1–3] or the use
of chimeric proteins to promote/improve crystal packing [4–8],
which has led to structures of important targets. However, less
effort has been applied towards the discovery, evaluation, and
optimization of crystals and nanocrystals. Here, we present the
use of transmission electron microscopy (TEM) as an easy, reproducible technique to guide the optimization of protein crystallization [9–12]. We will describe reproducible protocols to use TEM
for visualizing lattices from microcrystals, as well as fragmented
larger crystals and to study details of the crystallization process.
Our experiments have shown that for all protein crystals tested,
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_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
299
Detection of Microcrystals for CryoEM
Simon Weiss, Sandra Vergara, Guowu Lin, and Guillermo Calero
Abstract
Here, we present a strategy to identify microcrystals from initial protein crystallization screen experiments
and to optimize diffraction quality of those crystals using negative stain transmission electron microscopy
(TEM) as a guiding technique. The use of negative stain TEM allows visualization along the process and
thus enables optimization of crystal diffraction by monitoring the lattice quality of crystallization conditions. Nanocrystals bearing perfect lattices are seeded and can be used for MicroED as well as growing
larger crystals for X-ray and free electron laser (FEL) data collection.
Key words Protein crystallization, Optimization, Brightfield microscopy, UV microscopy, Negative
staining TEM, FFT-calculation, Granular aggregate, Microcrystal, Crystal lattice quality
1 Introduction
Crystallization of protein targets remains the most significant challenge in the process of structure determination by macromolecular
crystallography. When setting up crystallization trays experimenters
can obtain three different negative outcomes: (a) conditions with
amorphous precipitate, (b) few or no conditions with apparent
crystals or (c) crystals that do not diffract or diffract to low resolution. To tackle these problems most recent efforts have focused
mainly on improving sample “crystallizability” by implementing
techniques such as alanine scanning mutagenesis [1–3] or the use
of chimeric proteins to promote/improve crystal packing [4–8],
which has led to structures of important targets. However, less
effort has been applied towards the discovery, evaluation, and
optimization of crystals and nanocrystals. Here, we present the
use of transmission electron microscopy (TEM) as an easy, reproducible technique to guide the optimization of protein crystallization [9–12]. We will describe reproducible protocols to use TEM
for visualizing lattices from microcrystals, as well as fragmented
larger crystals and to study details of the crystallization process.
Our experiments have shown that for all protein crystals tested,
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_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
299