Chapter 10
2D Electron Crystallography of Membrane Protein Single-,
Double-, and Multi-Layered Ordered Arrays
Matthew C. Johnson, Yusuf M. Uddin, Kasahun Neselu, and
Ingeborg Schmidt-Krey
Abstract
The electron cryo-microscopy (cryo-EM) approach of 2D electron crystallography allows for structure
determination of two-dimensional (2D) crystals of soluble and membrane proteins, employing identical
principles and methods once 2D crystals are obtained. Two-dimensional crystallization trials of membrane
proteins can result in multiple outcomes of ordered arrays, which may be suited for either 2D electron
crystallography, helical analysis, or MicroED.
The membrane protein 2D crystals used for 2D electron crystallography are either single- or doublelayered ordered proteoliposome vesicles or sheet-like membranes. We have developed a cryo-EM grid
preparation approach, which allows for the analysis of stacked 2D crystals that are neither suitable for
MicroED nor for directly applying 2D electron crystallography. This new grid preparation approach, the
peel-blot, uses the capillary force generated by submicron filter paper and mechanical means for the
separation of stacked 2D crystals into single-layered 2D crystals, for which standard 2D electron crystallography can then be employed. The preparation of 2D crystals, the peel-blot grid preparation, and the
structure determination by 2D electron crystallography are described here.
Key words Membrane protein, Structure, Reconstitution, 2D Crystallization, Peel-blot, Cryo-EM,
2D Electron crystallography, 2D Crystallography, Electron crystallography
1 Introduction
The cryo-EM method of 2D electron crystallography provided the
first understanding of membrane protein secondary structure [1]
and resulted in some of the first atomic models of both membrane
proteins [2, 3] and soluble α/β tubulin protein [4, 5]. Just as
importantly, 2D electron crystallography allows for structure–function studies and has provided unprecedented resolution of membrane protein structure with detailed structural insights on the
entire surrounding phospholipid bilayer [6].
Following the “resolution revolution” [7] of cryo-EM,
subsequent ongoing development in single particle cryo-EM in
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_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
227
2D Electron Crystallography of Membrane Protein Single-,
Double-, and Multi-Layered Ordered Arrays
Matthew C. Johnson, Yusuf M. Uddin, Kasahun Neselu, and
Ingeborg Schmidt-Krey
Abstract
The electron cryo-microscopy (cryo-EM) approach of 2D electron crystallography allows for structure
determination of two-dimensional (2D) crystals of soluble and membrane proteins, employing identical
principles and methods once 2D crystals are obtained. Two-dimensional crystallization trials of membrane
proteins can result in multiple outcomes of ordered arrays, which may be suited for either 2D electron
crystallography, helical analysis, or MicroED.
The membrane protein 2D crystals used for 2D electron crystallography are either single- or doublelayered ordered proteoliposome vesicles or sheet-like membranes. We have developed a cryo-EM grid
preparation approach, which allows for the analysis of stacked 2D crystals that are neither suitable for
MicroED nor for directly applying 2D electron crystallography. This new grid preparation approach, the
peel-blot, uses the capillary force generated by submicron filter paper and mechanical means for the
separation of stacked 2D crystals into single-layered 2D crystals, for which standard 2D electron crystallography can then be employed. The preparation of 2D crystals, the peel-blot grid preparation, and the
structure determination by 2D electron crystallography are described here.
Key words Membrane protein, Structure, Reconstitution, 2D Crystallization, Peel-blot, Cryo-EM,
2D Electron crystallography, 2D Crystallography, Electron crystallography
1 Introduction
The cryo-EM method of 2D electron crystallography provided the
first understanding of membrane protein secondary structure [1]
and resulted in some of the first atomic models of both membrane
proteins [2, 3] and soluble α/β tubulin protein [4, 5]. Just as
importantly, 2D electron crystallography allows for structure–function studies and has provided unprecedented resolution of membrane protein structure with detailed structural insights on the
entire surrounding phospholipid bilayer [6].
Following the “resolution revolution” [7] of cryo-EM,
subsequent ongoing development in single particle cryo-EM in
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_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
227