Overview of Electron Crystallography of Membrane Proteins: Crystallization and Screening Strategies Using Negative Stain Electron Microscopy

Brent L. Nannenga1, Matthew G. Iadanza1, Breanna S. Vollmar1, Tamir Gonen2

1 Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, 2 Corresponding author
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 17.15
DOI:  10.1002/0471140864.ps1715s72
Online Posting Date:  April, 2013
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Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three‐dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X‐ray crystallography. Electron crystallography employs two‐dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well‐ordered two‐dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well‐ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution. Curr. Protoc. Protein Sci. 72:17.15.1‐17.15.11. © 2013 by John Wiley & Sons, Inc.

Keywords: electron crystallography; membrane proteins; negative stain electron microscopy; protein purification; protein solubilization

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Table of Contents

  • Introduction
  • Large‐Scale Production of Membrane Proteins
  • Membrane Protein Solubilization
  • Membrane Protein Purification
  • The Growth of 2D Crystals—Membrane Protein Reconstitution
  • Negative Staining EM as a Tool for Screening 2D Crystals
  • Conclusions
  • Acknowledgements
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
   Baneyx, F. 1999. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10:411‐421.
   Frey, T.G., Chan, S.H.P., and Schatz, G. 1978. Structure and orientation of cytochrome c oxidase in crystalline membranes. J. Biol. Chem. 253:4389‐4395.
   Fujiyoshi, Y. 1998. The structural study of membrane proteins by electron crystallography. Adv. Biophys. 35:25‐80.
   Gonen, T., Donaldson, P., and Kistler, J. 2000. Galectin‐3 is associated with the plasma membrane of lens fiber cells. Invest. Ophthalmol. Vis. Sci. 41:199‐203.
   Gonen, T., Sliz, P., Kistler, J., Cheng, Y., and Walz, T. 2004. Aquaporin‐0 membrane junctions reveal the structure of a closed water pore. Nature 429:193‐197.
   Gonen, T., Cheng, Y., Sliz, P., Hiroaki, Y., Fujiyoshi, Y., Harrison, S.C., and Walz, T. 2005. Lipid‐protein interactions in double‐layered two‐dimensional AQP0 crystals. Nature 438:633‐638.
   Hochuli, E. 1988. Large‐scale chromatography of recombinant proteins. J. Chromatogr. 444:293‐302.
   le Maire, M., Champeil, P., and Moller, J.V. 2000. Interaction of membrane proteins and lipids with solubilizing detergents. Biochim. Biophys. Acta 1508:86‐111.
   Lotz, M., Haase, W., Kuhlbrandt, W., and Collinson, I. 2008. Projection structure of yidC: A conserved mediator of membrane protein assembly. J. Mol. Biol. 375:901‐907.
   Mason, A.B., He, Q.Y., Halbrooks, P.J., Everse, S.J., Gumerov, D.R., Kaltashov, I.A., Smith, V.C., Hewitt, J., and MacGillivray, R.T. 2002. Differential effect of a his tag at the N‐ and C‐termini: Functional studies with recombinant human serum transferrin. Biochemistry 41:9448‐9454.
   Midgett, C.R. and Madden, D.R. 2007. Breaking the bottleneck: Eukaryotic membrane protein expression for high‐resolution structural studies. J. Struct. Biol. 160:265‐274.
   Miroux, B. and Walker, J.E. 1996. Over‐production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260:289‐298.
   Murata, K., Misuoka, K., Hirai, T., Walz, T., Agre, P., Heymann, J.B., Engel, A., and Fujiyoshi, Y. 2000. Structural determinants of water permeation through aquaporin‐1. Nature 407:599‐605.
   Nannenga, B.L. and Baneyx, F. 2011. Reprogramming chaperone pathways to improve membrane protein expression in Escherichia coli. Protein Sci. 20:1411‐1420.
   Ramon, A. and Marin, M. 2011. Advances in the production of membrane proteins in Pichia pastoris. Biotechnol. J. 6:700‐706.
   Reichow, S.L. and Gonen, T. 2009. Lipid‐protein interactions probed by electron crystallography. Curr. Opin. Struct. Biol. 19:560‐565.
   Rémigy, H.W., Caujolle‐Bert, D. Suda, K., Schenk, A., Chami, M., and Engel, A. 2003. Membrane protein reconstitution and crystallization by controlled dilution. FEBS Lett. 555:160‐169.
   Rigaud, J.L., Mosser, G., Lacaperre, J.J., Olofsson, A., Levy, D., and Ranck, J.L. 1997. Bio‐Beads: An efficient strategy for two‐dimensional crystallization of membrane proteins. J. Struct. Biol. 118:226‐235.
   Sastry, M.S., Zhou, W., and Baneyx, F. 2009. Integrity of N‐ and C‐termini is important for E. coli Hsp31 chaperone activity. Protein Sci. 18:1439‐1447.
   Sazanov, L.A. and Walker, J.E. 2000. Cryo‐electron crystallography of two sub‐complexes of bovine complex I reveals the relationship between the membrane and peripheral arms. J. Mol. Biol. 302:455‐464.
   Signorell, G.A., Kaufmann, T.C., Kukulski, W., Engel, A., and Remigy, H.W. 2007. Controlled 2D crystallization of membrane proteins using methyl‐beta‐cyclodextrin. J. Struct. Biol. 157:321‐328.
   Surade, S., Klein, M., Stolt‐Bergner, P.C., Muenke, C., Roy, A., and Michel, H. 2006. Comparative analysis and “expression space” coverage of the production of prokaryotic membrane proteins for structural genomics. Protein Sci. 15:2178‐2189.
   Tucker, J. and Grisshammer, R. 1996. Purification of a rat neurotensin receptor expressed in Escherichia coli. Biochem. J. 317:891‐899.
   Unwin, N. 2005. Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J. Mol. Biol. 346:967‐989.
   Viadiu, H., Gonen, T., and Walz, T. 2007. Projection map of aquaporin‐9 at 7 A resolution. J. Mol. Biol. 367:80‐88.
   Wagner, S., Klepsch, M.M., Schlegel, S., Appel, A., Draheim, R., Tarry, M., Högbom, M., van Wijk, K.J., Slotboom, D.J., Persson, J.O., and de Gier, J.W. 2008. Tuning Escherichia coli for membrane protein overexpression. Proc. Natl. Acad. Sci. U.S.A. 105:14371‐14376.
   Weiss, H.M. and Grisshammer, R. 2002. Purification and characterization of the human adenosine A(2a) receptor functionally expressed in Escherichia coli. Eur. J. Biochem. 269:82‐92.
   Williams, K.A., Geldmacher‐Kaufer, U., Padan, E., Schuldiner, S., and Kuhlbrandt, W. 1999. Projection structure of NhaA, a secondary transporter from Escherichia coli, at 4.0 A resolution. EMBO J. 18:3558‐3563.
   Wisedchaisri, G., Reichow, S.L., and Gonen, T. 2011. Advances in structural and functional analysis of membrane proteins by electron crystallography. Structure 19:1381‐1393.
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