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High-Throughput Methods for Electron Crystallography

  • David L. Stokes
  • Iban Ubarretxena-Belandia
  • Tamir Gonen
  • Andreas Engel
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 955)

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 

Notes

Acknowledgments

The authors would like to acknowledge a number of individuals who have developed the methodologies discussed in this chapter. In particular, Martin Vink, Changki Kim, and Minghui Hu, Thomas Kaufmann, Ioan Iacovache, Nicolas Coudray Hervé Rémigy and many others were responsible for designing and implementing the devices and protocols described in this chapter. The authors are indebted to them for their efforts. The authors belong to the Transcontinental EM Initiative for Membrane Protein Structure, which is a center for membrane protein structure determination funded by the NIH Protein Structure Initiative under grant U54GM094598. More information about this center can be found at http://temimps.nysbc.org. Additional research support was provided by NIH grants R01GM081817, R01GM095747, and R01GM079233.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • David L. Stokes
    • 1
  • Iban Ubarretxena-Belandia
    • 2
  • Tamir Gonen
    • 3
  • Andreas Engel
    • 4
  1. 1.Department of Cell Biology, Skirball Institute of Biomolecular MedicineNew York University School of MedicineNew YorkUSA
  2. 2.Department of Structural and Chemical BiologyMt. Sinai School of MedicineNew YorkUSA
  3. 3.Janelia Farm Research Campus, Howard Hughes Medical InstituteAshburnUSA
  4. 4.Department of PharmacologyCase Western Reserve UniversityClevelandUSA

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