Chapter 11
Sample Preparation and Data Collection for Electron
Crystallographic Studies on Membrane Protein Structures
and Lipid–Protein Interaction
Ka-Yi Chan, Chloe Du Truong, Yu-Ping Poh, and Po-Lin Chiu
Abstract
Electron crystallography is a unique tool to study membrane protein structures and lipid–protein interactions in their native-like environments. Two-dimensional (2D) protein crystallization enables the lipids
immobilized by the proteins, and the generated high-resolution density map allows us to model the atomic
coordinates of the surrounding lipids to study lipid–protein interaction. This protocol describes the sample
preparation for electron crystallographic studies, including back-injection method and carbon sandwich
method. The protocols of data collection for electron crystallography, including electron imaging and
diffraction, of the 2D membrane crystal will be followed.
Key words Electron crystallography, 2D Crystals, Sugar embedding, Back-injection method, Carbon-sandwich method, Electron diffraction
1 Introduction
Membrane proteins are the proteins residing in biological membranes. In most organisms, about 20–30% of the genes encode
membrane proteins [1, 2], which play important roles as a gatekeeper, a signal transducer, or a diode in the membranes for living
processes of a cell [3]. They serve as the main targets for drug
design in promotion or inhibition of a signaling transduction pathway or modulation of the ion channel conductance [4]. Thus, it is
essential to understand the structures of these proteins and how
they coordinate with their surrounding proteins or lipids to synergistically perform their function. Two-dimensional (2D) protein
crystallization combined with electron cryogenic microscopy
(cryo-EM), particularly electron crystallography, is one of the powerful techniques that are used to determine the protein structures in
the membrane, which is in their native-like environment. A 2D
protein array of the membrane protein is crystallized with lipids
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_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021
247
Sample Preparation and Data Collection for Electron
Crystallographic Studies on Membrane Protein Structures
and Lipid–Protein Interaction
Ka-Yi Chan, Chloe Du Truong, Yu-Ping Poh, and Po-Lin Chiu
Abstract
Electron crystallography is a unique tool to study membrane protein structures and lipid–protein interactions in their native-like environments. Two-dimensional (2D) protein crystallization enables the lipids
immobilized by the proteins, and the generated high-resolution density map allows us to model the atomic
coordinates of the surrounding lipids to study lipid–protein interaction. This protocol describes the sample
preparation for electron crystallographic studies, including back-injection method and carbon sandwich
method. The protocols of data collection for electron crystallography, including electron imaging and
diffraction, of the 2D membrane crystal will be followed.
Key words Electron crystallography, 2D Crystals, Sugar embedding, Back-injection method, Carbon-sandwich method, Electron diffraction
1 Introduction
Membrane proteins are the proteins residing in biological membranes. In most organisms, about 20–30% of the genes encode
membrane proteins [1, 2], which play important roles as a gatekeeper, a signal transducer, or a diode in the membranes for living
processes of a cell [3]. They serve as the main targets for drug
design in promotion or inhibition of a signaling transduction pathway or modulation of the ion channel conductance [4]. Thus, it is
essential to understand the structures of these proteins and how
they coordinate with their surrounding proteins or lipids to synergistically perform their function. Two-dimensional (2D) protein
crystallization combined with electron cryogenic microscopy
(cryo-EM), particularly electron crystallography, is one of the powerful techniques that are used to determine the protein structures in
the membrane, which is in their native-like environment. A 2D
protein array of the membrane protein is crystallized with lipids
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_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021
247