Journal of Structural Biology 151 (2005) 288–297 www.elsevier.com/locate/yjsbi 1047-8477/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2005.07.004 A “Xip–Xop” rotation stage for routine dual-axis electron cryotomography Cristina V. Iancu a,1 , Elizabeth R. Wright a,1 , Jordan Benjamin a , William F. Tivol a , D. Prabha Dias a , Gavin E. Murphy a , Robert C. Morrison b , J. Bernard Heymann c , Grant J. Jensen a,¤ a Division of Biology, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125, USA b Gatan (UK), 25 NuYeld Way, Abingdon Oxon OX14 1RL, UK c Laboratory of Structural Biology Research, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA Received 3 March 2005; received in revised form 6 June 2005; accepted 6 July 2005 Available online 11 August 2005 Abstract 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 “Xip–Xop” 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.  2005 Elsevier Inc. All rights reserved. Keywords: Tomography; Electron microscopy; cryoEM; Dual-axis; Cryoholder; Missing wedge 1. Introduction The highest resolution technique currently available for three-dimensional structural studies of unique objects is electron tomography, in which a specimen is imaged multiple times in an electron microscope (EM) while being incrementally tilted through a range of views. The method has the potential to couple the exqui- site spatial resolution of modern electron microscopes, which in some cases can now be even sub-Angstrom (Batson et al., 2002; Hosokawa et al., 2003), with three- dimensional structure determination through a variety of reconstruction algorithms. Thus, electron tomogra- phy is emerging as a powerful new technique in both materials and life science research (Baumeister, 2004; Subramaniam and Milne, 2004; Ziese et al., 2004). There are several practical limitations for biological samples, however, including radiation damage and the typically restricted range of tilt-angles from which images can be recorded. The tilt-angle limitation arises because most EM samples are thin disks of material approximately 3 mm in diameter and »30–500 nm thick, and as these samples are incrementally tilted, the depth of material the electron beam must pass through increases as one over the cosine of the tilt-angle. At high tilt-angles, most samples become prohibitively thick. * Corresponding author. Fax: +1 626 395 5730. E-mail address: Jensen@caltech.edu (G.J. Jensen). 1 These authors contributed equally.