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Electron Tomography of Paracrystalline 2D Arrays

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Electron Crystallography of Soluble and Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 955))

Abstract

Paracrystalline arrays possess specific types of disorder that reduce the structural information as well as resolution when spatially averaged over repeating motifs. Electron tomography combined with motif classification and averaging can solve the heterogeneity problem and provide information on the structural elements that give rise to the disorder. This chapter describes procedures that would be used in a typical tomography application to identify and characterize a paracrystalline specimen. Particular emphasis is given to actively contracting insect flight muscle, a specimen with particularly difficult to characterize structural heterogeneity and 2D paracrystalline arrays of myosin-V, from which a particularly high resolution motif average was obtained. All aspects of the study are described including data collection, merging of micrographs to produce the tomogram, alignment to an invariant structural element, classification and averaging of heterogeneous structures, and reassembly of focused class averages into high signal-to-noise ratio representations of the original raw repeats. Particular emphasis is placed on limitations of the various processes to produce the final class averages.

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Acknowledgments

The research on insect flight muscle was supported by NIH Grant GM30598, the myosin-V reconstruction under NIH grant AR47421, and protomo is being developed under NIH grant GM82948.

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Authors and Affiliations

  1. Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, USA

    Hanspeter Winkler & Kenneth A. Taylor

  2. Department of Biochemistry & Biophysics, University of California San Francisco, San Francisco, CA, USA

    Shenping Wu

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  1. Hanspeter Winkler
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  2. Shenping Wu
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  3. Kenneth A. Taylor
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Correspondence to Kenneth A. Taylor .

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Editors and Affiliations

  1. School of Biology, Georgia Institute of Technology, Ferst Dr. 310, Atlanta, 30332, Georgia, USA

    Ingeborg Schmidt-Krey

  2. , Dept. Biochemistry & Biophysics, University of California, San Francisco, 16th St. 600, San Francisco, 94158, California, USA

    Yifan Cheng

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Winkler, H., Wu, S., Taylor, K.A. (2013). Electron Tomography of Paracrystalline 2D Arrays. 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_23

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  • DOI: https://doi.org/10.1007/978-1-62703-176-9_23

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