A "flip-flop" rotation stage for routine dual-axis electron cryotomography

J Struct Biol. 2005 Sep;151(3):288-97. doi: 10.1016/j.jsb.2005.07.004.

Abstract

Electron cryotomography can be used to solve the three-dimensional structures of individual large macromolecules, assemblies, and even small intact cells to medium (approximately 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 degrees 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cryoelectron Microscopy / instrumentation*
  • Cryoelectron Microscopy / methods
  • HIV-1 / ultrastructure
  • Humans
  • Image Processing, Computer-Assisted
  • Imaging, Three-Dimensional
  • Prokaryotic Cells / ultrastructure
  • Tomography*