Two-Dimensional Crystallization of Integral Membrane Proteins for Electron Crystallography
Although membrane proteins make up 30% of the proteome and are a common target for therapeutic drugs, determination of their atomic structure remains a technical challenge. Electron crystallography represents an alternative to the conventional methods of
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1. Introduction Electron crystallography is a method of membrane protein structure determination that involves imaging two-dimensional (2D) crystals by cryoelectron microscopy (cryo-EM). Such 2D crystals are produced when membrane proteins adopt a regular array within the plane of a lipid bilayer. Methods for electron crystallography were originally pioneered in the 1970s by Henderson and Unwin in their studies of bacteriorhodopsin (1). Intense efforts over the next two decades led to developments both in electron microscope design and in software for analyzing the resulting images, thus producing the first atomic resolution structure of a membrane protein in its native membranous Jean-Jacques Lacapère (ed.), Membrane Protein Structure Determination: Methods and Protocols, Methods in Molecular Biology, vol. 654, DOI 10.1007/978-1-60761-762-4_10, © Springer Science+Business Media, LLC 2010
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environment (2). Since then, electron crystallography has developed into a powerful tool to elucidate the 3D structure of a wide range of membrane proteins (3–6), thus offering a plausible alternative to X-ray crystallography. Similar to X-ray crystallography, the bottleneck preventing a more generalized use of electron crystallography is the preparation of well-ordered crystals. In the case of membrane proteins, there are three predominant morphologies adopted by 2D crystals: (1) flattened lipid vesicles with two, overlapping 2D lattices; (2) tubular lipid vesicles which retain a cylindrical shape and comprise a helical array of membrane proteins; and (3) a planar lipid bilayer with a single, coherent 2D array of proteins. Historically, 2D crystallization of membrane proteins has involved either in situ crystallization or in vitro reconstitution (7, 8). The advantage of in situ crystallization is that the membrane protein is never removed from its native membrane. However, this approach requires a high concentration of the relevant protein in the native membrane and is not therefore generally applicable. More generally, 2D crystals have been grown by reconstitution of purified, detergent-solubilized membrane proteins into lipid bilayers under defined conditions (7, 9). Reconstitution involves the controlled removal of detergent, either by dialysis or by adsorption to a hydrophobic resin, in the presence of exogenous lipid. Under favorable conditions, the membrane proteins assemble into an ordered 2D array within the resulting lipid bilayers (7). In this chapter, we will describe protocols and procedures to obtain 2D crystals of membrane proteins and to evaluate their quality and potential for structure determination by electron crystallography.
2. Materials 2.1. Analysis of Lipid
1. Solid phase: 20 × 20 cm thin layer chromatography (TLC) plates with silica gel 60 F254 coating (Merck & Co., Whitehouse Station, NJ). 2. Glass cutter. 3. Glass chromatography tank (25 cm × 27 cm × 10 cm). 4. Graduated disposable 5 ml glass capillaries (CAMAG, Muttenz, Switzerland). 5. Mobile phase: mixture of chloroform/
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