Chapter 12
Single Particle Analysis for High-Resolution 2D Electron
Crystallography
Ricardo Righetto and Henning Stahlberg
Abstract
Electron crystallography has been used for decades to determine three-dimensional structures of membrane
proteins embedded in a lipid bilayer. However, high-resolution information could only be retrieved from
samples where the 2D crystals were well ordered and perfectly flat. This is rarely the case in practice. We
implemented in the FOCUS package a module to export transmission electron microscopy images of 2D
crystals for 3D reconstruction by single particle algorithms. This approach allows for correcting local
distortions of the 2D crystals, yielding much higher resolution reconstructions than otherwise expected
from the observable diffraction spots. In addition, the single particle framework enables classification of
heterogeneous structures coexisting within the 2D crystals. We provide here a detailed guide on single
particle analysis of 2D crystal data based on the FOCUS and FREALIGN packages.
Key words Electron crystallography, 2D Crystals, Single particle analysis, Membrane proteins,
MloK1, Ion channels, Cryo-electron microscopy, Image processing, 3D Reconstruction, 3D
Classification
1 Introduction
Historically, the structures of membrane proteins have been much
more difficult to determine than those of their soluble counterparts. This is mainly due to the hydrophobic nature of the transmembrane domains, which render these proteins challenging both
for crystallization and solubilization. However, a special type of
crystallography, in which the proteins form ordered
two-dimensional (2D) periodical arrays in a lipid membrane, is
suitable for gathering information on their three-dimensional
(3D) structures. It was from a naturally occurring 2D crystal, the
purple membrane of Halobacterium salobium, that the first 3D
structure of a membrane protein was obtained [1]. This breakthrough made the transmission electron microscope (TEM) a
promising instrument for studying membrane proteins, as long as
2D crystals could be obtained. In fact, after it was demonstrated
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_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
267
Single Particle Analysis for High-Resolution 2D Electron
Crystallography
Ricardo Righetto and Henning Stahlberg
Abstract
Electron crystallography has been used for decades to determine three-dimensional structures of membrane
proteins embedded in a lipid bilayer. However, high-resolution information could only be retrieved from
samples where the 2D crystals were well ordered and perfectly flat. This is rarely the case in practice. We
implemented in the FOCUS package a module to export transmission electron microscopy images of 2D
crystals for 3D reconstruction by single particle algorithms. This approach allows for correcting local
distortions of the 2D crystals, yielding much higher resolution reconstructions than otherwise expected
from the observable diffraction spots. In addition, the single particle framework enables classification of
heterogeneous structures coexisting within the 2D crystals. We provide here a detailed guide on single
particle analysis of 2D crystal data based on the FOCUS and FREALIGN packages.
Key words Electron crystallography, 2D Crystals, Single particle analysis, Membrane proteins,
MloK1, Ion channels, Cryo-electron microscopy, Image processing, 3D Reconstruction, 3D
Classification
1 Introduction
Historically, the structures of membrane proteins have been much
more difficult to determine than those of their soluble counterparts. This is mainly due to the hydrophobic nature of the transmembrane domains, which render these proteins challenging both
for crystallization and solubilization. However, a special type of
crystallography, in which the proteins form ordered
two-dimensional (2D) periodical arrays in a lipid membrane, is
suitable for gathering information on their three-dimensional
(3D) structures. It was from a naturally occurring 2D crystal, the
purple membrane of Halobacterium salobium, that the first 3D
structure of a membrane protein was obtained [1]. This breakthrough made the transmission electron microscope (TEM) a
promising instrument for studying membrane proteins, as long as
2D crystals could be obtained. In fact, after it was demonstrated
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_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
267