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Electron Tomography of Paracrystalline 2D Arrays

  • Hanspeter Winkler
  • Shenping Wu
  • Kenneth A. Taylor
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Cryoelectron microscopy Electron microscope tomography Computer-assisted image processing Three-dimensional imaging 

Notes

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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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Hanspeter Winkler
    • 1
  • Shenping Wu
    • 2
  • Kenneth A. Taylor
    • 1
  1. 1.Institute of Molecular BiophysicsFlorida State UniversityTallahasseeUSA
  2. 2.Department of Biochemistry & BiophysicsUniversity of California San FranciscoSan FranciscoUSA

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