Iancu, Cristina V. and Wright, Elizabeth R. and Benjamin, Jordan and Tivol, William F. and Dias, D. Prabha and Murphy, Gavin E. and Morrison, Robert C. and Heymann, J. Bernard and Jensen, Grant J. (2005) A “flip–flop” rotation stage for routine dual-axis electron cryotomography. Journal of Structural Biology, 151 (3). pp. 288-297. ISSN 1047-8477. http://resolver.caltech.edu/CaltechAUTHORS:20110803-105303474
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Electron cryotomography can be used to solve the three-dimensional structures of individual large macromolecules, assemblies, and even small intact cells to medium (~4–8 nm) resolution in a near-native state, but restrictions in the range of accessible views are a major limitation. Here we report on the design, characterization, and demonstration of a new “flip–flop” rotation stage that allows facile and routine collection of two orthogonal tilt-series of cryosamples. Single- and dual-axis tomograms of a variety of samples are compared to illustrate qualitatively the improvement produced by inclusion of the second tilt-series. Exact quantitative expressions are derived for the volume of the remaining “missing pyramid” in reciprocal space. When orthogonal tilt-series are recorded to ±65° in each direction, as this new cryostage permits, only 11% of reciprocal space is left unmeasured. The tomograms suggest that further improvement could be realized, however, through better software to align and merge dual-axis tilt-series of cryosamples.
|Additional Information:||Copyright © 2005 Elsevier Inc. Received 3 March 2005; revised 6 June 2005; accepted 6 July 2005. Available online 11 August 2005. We thank D. Mastronarde for assistance with IMOD, H.J. Ding and B. Wen for image processing, A. Martino for providing purified carboxysomes, W. Sundquist for purified HIV-1 virus-like particles, and S. Tivol for reading the manuscript. This work was supported in part by NIH Grant PO1 GM66521 to G.J.J., DOE Grant DE-FG02-04ER63785 to G.J.J., the Beckman Institute at Caltech, and gifts to Caltech from the Ralph M. Parsons Foundation, the Agouron Institute, and the Gordon and Betty Moore Foundation.|
|Subject Keywords:||Tomography; Electron microscopy; cryoEM; Dual-axis; Cryoholder; Missing wedge|
|Official Citation:||Cristina V. Iancu, Elizabeth R. Wright, Jordan Benjamin, William F. Tivol, D. Prabha Dias, Gavin E. Murphy, Robert C. Morrison, J. Bernard Heymann, Grant J. Jensen, A 'flip-flop' rotation stage for routine dual-axis electron cryotomography, Journal of Structural Biology, Volume 151, Issue 3, September 2005, Pages 288-297, ISSN 1047-8477, DOI: 10.1016/j.jsb.2005.07.004. (http://www.sciencedirect.com/science/article/pii/S1047847705001437)|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||George Porter|
|Deposited On:||03 Aug 2011 18:05|
|Last Modified:||03 Aug 2011 18:05|
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