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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Ubarretxena-Belandia I, Stokes DL (2010) Present and future of membrane protein structure determination by electron crystallography. Adv Protein Chem Struct Biol 81:33–60
Schenk AD, Castano-Diez D, Gipson B, Arheit M, Zeng X, Stahlberg H (2010) 3D reconstruction from 2D crystal image and diffraction data. Methods Enzymol 482:101–129
Hite RK, Raunser S, Walz T (2007) Revival of electron crystallography. Curr Opin Struct Biol 17:389–395
Abeyrathne PD, Chami M, Pantelic RS, Goldie KN, Stahlberg H (2010) Preparation of 2D crystals of membrane proteins for high-resolution electron crystallography data collection. Methods Enzymol 481:25–43
White SH (2009) Biophysical dissection of membrane proteins. Nature 459:344–346
Luft JR, Collins RJ, Fehrman NA, Lauricella AM, Veatch CK, DeTitta GT (2003) A deliberate approach to screening for initial crystallization conditions of biological macromolecules. J Struct Biol 142:170–179
Rees DC (2001) Crystallographic analyses of hyperthermophilic proteins. Methods Enzymol 334:423–437
Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51
Nickell S, Forster F, Linaroudis A, Net WD, Beck F, Hegerl R, Baumeister W, Plitzko JM (2005) TOM software toolbox: acquisition and analysis for electron tomography. J Struct Biol 149:227–234
Suloway C, Shi J, Cheng A, Pulokas J, Carragher B, Potter CS, Zheng SQ, Agard DA, Jensen GJ (2009) Fully automated, sequential tilt-series acquisition with Leginon. J Struct Biol 167:11–18
Zheng SQ, Keszthelyi B, Branlund E, Lyle JM, Braunfeld MB, Sedat JW, Agard DA (2007) UCSF tomography: an integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction. J Struct Biol 157:138–147
Koster AJ, Chen H, Sedat JW, Agard DA (1992) Automated microscopy for electron tomography. Ultramicroscopy 46:207–227
Suloway C, Pulokas J, Fellmann D, Cheng A, Guerra F, Quispe J, Stagg S, Potter CS, Carragher B (2005) Automated molecular microscopy: the new Leginon system. J Struct Biol 151:41–60
Zhang J, Nakamura N, Shimizu Y, Liang N, Liu X, Jakana J, Marsh MP, Booth CR, Shinkawa T, Nakata M, Chiu W (2009) JADAS: a customizable automated data acquisition system and its application to ice-embedded single particles. J Struct Biol 165:1–9
Zhang P, Beatty A, Milne JL, Subramaniam S (2001) Automated data collection with a Tecnai 12 electron microscope: applications for molecular imaging by cryomicroscopy. J Struct Biol 135:251–261
Oostergetel GT, Keegstra W, Brisson A (1998) Automation of specimen selection and data acquisition for protein crystallography. Ultramicroscopy 74:47–50
Cheng A, Leung A, Fellmann D, Quispe J, Suloway C, Pulokas J, Abeyrathne PD, Lam JS, Carragher B, Potter CS (2007) Towards automated screening of two-dimensional crystals. J Struct Biol 160:324–331
Hu M, Vink M, Kim C, Derr K, Koss J, D’Amico K, Ubarretxena-Belandia I, Stokes DL (2010) Automated electron microscopy for evaluating two-dimensional crystallization of membrane proteins. J Struct Biol 171(1):102–110
Coudray N, Hermann G, Caujolle-Bert D, Karathanou A, Erne-Brand F, Buessler JL, Daum P, Plitzko JM, Chami M, Mueller U, Kihl H, Urban JP, Engel A, Remigy HW (2010) Automated screening of 2D crystallization trials using transmission electron microscopy: a high-throughput tool-chain for sample preparation and microscopic analysis. J Struct Biol 173(2):365–374
Kaufmann TC, Engel A, Remigy HW (2006) A novel method for detergent concentration determination. Biophys J 90:310–317
Vink M, Derr K, Love J, Stokes DL, Ubarretxena-Belandia I (2007) A high-throughput strategy to screen 2D crystallization trials of membrane proteins. J Struct Biol 160:295–304
Iacovache I, Biasini M, Kowal J, Kukulski W, Chami M, Van der Goot FG, Engel A, Remigy HW (2009) The 2DX robot: a membrane protein 2D crystallization Swiss Army knife. J Struct Biol 169(3):370–378
Remigy HW, Caujolle-Bert D, Suda K, Schenk A, Chami M, Engel A (2003) Membrane protein reconstitution and crystallization by controlled dilution. FEBS Lett 555:160–169
Rigaud J-L, Mosser G, Lacapere J-J, Olofsson A, Levy D, Ranck J-L (1997) Bio-beads: an efficient strategy for two-dimensional crystallization of membrane proteins. J Struct Biol 118:226–235
Signorell GA, Kaufmann TC, Kukulski W, Engel A, Remigy HW (2007) Controlled 2D crystallization of membrane proteins using methyl-beta-cyclodextrin. J Struct Biol 157:321–328
Kim C, Vink M, Hu M, Stokes DL, Ubarretxena-Belandia I (2010) An automated pipeline to screen membrane protein 2D crystallization. J Struct Funct Genomics 11(2):155–166
Lefman J, Morrison R, Subramaniam S (2007) Automated 100-position specimen loader and image acquisition system for transmission electron microscopy. J Struct Biol 158:318–326
Zolnai Z, Lee PT, Li J, Chapman MR, Newman CS, Phillips GN Jr, Rayment I, Ulrich EL, Volkman BF, Markley JL (2003) Project management system for structural and functional proteomics: Sesame. J Struct Funct Genomics 4:11–23
Haquin S, Oeuillet E, Pajon A, Harris M, Jones AT, van Tilbeurgh H, Markley JL, Zolnai Z, Poupon A (2008) Data management in structural genomics: an overview. Methods Mol Biol 426:49–79
Stokes DL, Rice WJ, Hu M, Kim C, Ubarretxena-Belandia I (2010) Two-dimensional crystallization of integral membrane proteins for electron crystallography. Methods Mol Biol 654:187–205
Gyobu N, Tani K, Hiroaki Y, Kamegawa A, Mitsuoka K, Fujiyoshi Y (2004) Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique. J Struct Biol 146:325–333
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Stokes, D.L., Ubarretxena-Belandia, I., Gonen, T., Engel, A. (2013). High-Throughput Methods for Electron Crystallography. In: Schmidt-Krey, I., Cheng, Y. (eds) Electron Crystallography of Soluble and Membrane Proteins. Methods in Molecular Biology, vol 955. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-176-9_15
Download citation
DOI: https://doi.org/10.1007/978-1-62703-176-9_15
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-175-2
Online ISBN: 978-1-62703-176-9
eBook Packages: Springer Protocols