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Electron Tomographic Studies of a Large Macromolecular Assembly Preserved In Vitreous Ice: The Yeast Spindle Pole Body Core

Advances in both electron cryomicroscopy and tomographic reconstruction methods have made it possible to collect and analyze data from large macromolecular assemblies frozen without fixation, sectioning, or staining. The Spindle Pole Body (SPB) is the microtubule organizing center in the yeast Saccharomyces, and is involved in mitosis, nuclear movement, budding, and mating. We have used electron cryomicroscopy and tomographic reconstruction to investigate the 1500Å diameter, 600Å high cylindrical core structure produced by limited heparin treatment of isolated SPBs. Tilt series' of SPB cores preserved in vitreous ice were taken under low electron dose conditions. These images have been analyzed using iterative correlation alignment and computed back projections for tilt data without fiducial markers. In these studies, electron micrographs were digitized, and translationally aligned using correlation methods.