A new twist on protein crystallization

Electron microscopy (EM), combined with image processing, has become an increasingly powerful tool for structural biology. Advances in cryopreservation and imaging, along with increasingly sophisticated computational tools for image processing, allow three-dimensional (3D) structure determination of macromolecules or macromolecular assemblies trapped in native states (1, 2). Structures from noncrystalline specimens of large macromolecular assemblies that are inaccessible to NMR and extremely challenging for x-ray crystallography can be determined to resolutions approaching 7 Å for highly symmetric structures such as icosahedral viruses (3, 4) or around 15 Å for asymmetric structures (5). Currently, for higher resolution information, ordered specimens are required to facilitate orientation and averaging of views. Crystalline specimens have generally come in two flavors, either very thin, usually single-layer thick or two-dimensional (2D), crystals, or helical assemblies. The largest impact of EM and image processing to date has come from the analysis of 2D crystals, which has sometimes yielded near atomic resolution structures of membrane proteins and tubulin, each presenting formidable problems for preparing suitable 3D crystals for x-ray analysis (6–10). A method specifically designed to generate 2D crystals of soluble macromolecules has been introduced, facilitating the application of EM and image processing to a wider range of systems (11–13). While helical specimens …