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Electron Crystallography of Soluble and Membrane Proteins

Volume 955 of the series Methods in Molecular Biology pp 273-296

Date:

High-Throughput Methods for Electron Crystallography

  • David L. StokesAffiliated withDepartment of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University School of Medicine Email author 
  • , Iban Ubarretxena-BelandiaAffiliated withDepartment of Structural and Chemical Biology, Mt. Sinai School of Medicine
  • , Tamir GonenAffiliated withJanelia Farm Research Campus, Howard Hughes Medical Institute
  • , Andreas EngelAffiliated withDepartment of Pharmacology, Case Western Reserve University

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Abstract

Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing a native lipid environment for these proteins. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, electron microscopy can be used to collect images and diffraction and the corresponding data can be combined to produce a three-dimensional reconstruction, which under favorable conditions can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on titration of cyclodextrin as a chelating agent for detergent; a specialized pipetting robot has been designed not only to add cyclodextrin in a systematic way, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described.

Key words

Electron crystallography Electron microscopy Membrane proteins Protein structure High-throughput Crystallization Dialysis Cyclodextrin Negative stain