A "flip-flop" rotation stage for routine dual-axis electron cryotomography.
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.
Iancu
Cristina V
CV
Division of Biology, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125, USA.
Wright
Elizabeth R
ER
Benjamin
Jordan
J
Tivol
William F
WF
Dias
D Prabha
DP
Murphy
Gavin E
GE
Morrison
Robert C
RC
Heymann
J Bernard
JB
Jensen
Grant J
GJ
D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods; D015497:HIV-1 / Q000648:ultrastructure; D006801:Humans; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D011387:Prokaryotic Cells / Q000648:ultrastructure; D014054:Tomography