Marker-Free Alignment of Dual-Axis Tilt Series and Subvolume Analysis of Data from Dual-Axis Tomograms Hanspeter Winkler and Kenneth A. Taylor Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306 USA In recent years, electron tomography has become a mainstream technique for 3D reconstruction of macro- molecules, macromolecular assemblies, organelles, and whole cells. While the resolution that can be obtained with tomography is not as high as that obtained by alternative techniques such as single particle methods, the analysis of subvolumes extracted from tomograms provides a relatively straightforward way to characterize structural or conformational heterogeneity with well established alignment and classi ca- tion techniques. One of the problems encountered in electron tomography is incomplete sampling of the reconstruction in Fourier space, which is due to the limited range in which the specimen can be tilted. Typical angular ranges from ±60° to ±75° leave a considerable volume unsampled: a wedge shaped region for single-axis tilt series, or a pyramidal region for dual-axis tilt series, respectively. Dual-axis tomography results in a better coverage of Fourier space and a more isotropic resolution, since only the intersection of the missing wedges of the two contributing single-axis tilt series with approximately perpendicular axes remains unsampled. The more complete coverage is also advantageous for the processing of subvolumes extracted from such tomograms. Computational means to compensate missing wedge effects in the cross-correlation alignment or the classi cation of subvolumes then become less critical in order to produce reliable averaged structures. Using dual-axis tilt series of a plastic embedded and sectioned insect flight muscle specimen, we have explored two approaches for tilt series alignment and 3D reconstruction: first, the separate processing of a dual-axis tilt series as two single-axis tilt series with subsequent merging of the two computed maps [7], and second, a novel simultaneous alignment technique [8]. The merging of the two tomograms in the rst approach was carried out according to Mastronarde [3]; the single map in the second approach was computed with weighted back-projection from the combined simultaneously aligned data. In both approaches we used marker-free tilt series alignment [4]. This procedure is based on area matching, in which the images are not only translationally aligned, but also matched by considering rotation, shear and scale changes. In addition, it includes a re nement of geometric parameters and a reference construction scheme based on the computation of a preliminary, continually evolving 3D reconstruction. Alignment is carried out with respect to computed references which are reprojected from the preliminary reconstruc- tion. The whole registration procedure is iterative, consisting of cycles of area matching and geometry re nement. For the subsequent subvolume processing and resolution assessment we applied procedures that we de- veloped for the analysis of cryo-tomograms of myosin V, SIV and HIV virions [6], that included missing wedge compensation. Subvolumes were extracted from the dual-axis tomograms at identical positions relative to the specimen structure for comparison purposes. The analysis showed that the simultaneous alignment approach produced averages with higher resolution as measured with Fourier shell correlation 766 doi:10.1017/S1431927613005825 Microsc. Microanal. 19 (Suppl 2), 2013 © Microscopy Society of America 2013 https://doi.org/10.1017/S1431927613005825 Downloaded from https://www.cambridge.org/core. IP address: 181.215.201.117, on 06 May 2020 at 00:45:16, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms.