title: A "flip-flop" rotation stage for routine dual-axis electron cryotomography.
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.
authors.affiliation: Division of Biology, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125, USA.
authors.forename: Cristina V
authors.initials: CV
authors.lastname: Iancu
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Wright
authors.forename: Jordan
authors.initials: J
authors.lastname: Benjamin
authors.forename: William F
authors.initials: WF
authors.lastname: Tivol
authors.forename: D Prabha
authors.initials: DP
authors.lastname: Dias
authors.forename: Gavin E
authors.initials: GE
authors.lastname: Murphy
authors.forename: Robert C
authors.initials: RC
authors.lastname: Morrison
authors.forename: J Bernard
authors.initials: JB
authors.lastname: Heymann
authors.forename: Grant J
authors.initials: GJ
authors.lastname: Jensen
mesh_terms: 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