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Use of Single Particle Methods to Reconstruct Helical Reca and F-Actin Filaments

Published online by Cambridge University Press:  02 July 2020

E.H. Egelman ,
A. Orlova  and
X. Yu
E.H. Egelman
Affiliation:
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA22908
A. Orlova
Affiliation:
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA22908
X. Yu
Affiliation:
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA22908
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Extract

RecA has been the most intensively studied enzyme in homologous genetic recombination. Actin is one of the most abundant and conserved eukaryotic proteins, and is central to muscle contraction, cell motility, and the control of cell shape. Both form helical filaments, and we have been studying both for many years, primarily using negatively stained specimens. We and others have used cryo-EM of frozen-hydrated RecA and F-actin samples, but the resolution obtained by this method is actually worse than that obtained from negatively stained specimens. The criterion for resolution that we have used is the match with an atomic model for F-actin, since this is a much better standard than statistical measures such as Fourier ring correlations or phase residuals, which can only measure the internal consistency of the data.

F-actin is a flexible structure, both with respect to bending and torsional motions of subunits. The RecA filament is 10 times more flexible than F-actin. The bending of RecA and F-actin require that images be straightened, and the ability of a user to fit a spline function to the filament axis limits the precision of the method. The method is only valid for small perturbations from a rigid rod, and thus will introduce artifacts for real filaments. In order to straighten filament images obtained in ice, one needs sufficient contrast (obtained by a large defocus) to be able to apply such algorithms. The torsional motions of the actin subunits, which may be as large as 5-6° per subunit, limit the resolution that can be obtained as one averages over long lengths.

Type
Cryotechniques, Immunocytochemistry, and Electron Microscopy I. Molecular Approach
Information
Microscopy and Microanalysis , Volume 5 , Issue S2: Proceedings: Microscopy & Microanalysis '99, Microscopy Society of America 57th Annual Meeting, Microbeam Analysis Society 33rd Annual Meeting, Portland, Oregon August 1-5, 1999 , August 1999 , pp. 404 - 405
Copyright
Copyright © Microscopy Society of America

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References

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