Visualization of β-sheets and some amino acid side chains in 400 kV electron images of streptavidin crystallized on phospholipid monolayers

Abstract

Two-dimensional (2-D) protein crystallization on lipid monolayers is an emerging technique with potential application to structure elucidation of biological macromolecules (Uzgiris and Kornberg, 1983). Among more than a dozen proteins or macromolecular assemblies which have been crystallized by this technique, streptavidin 2-D crystals diffract electrons beyond 3 Å (Kubalek, et al., 1991), indicating that structural information from them is retrievable to near-atomic resolution. This 15 kD protein, synthesized by Streptomyces avidinii, is probably the single most widely used protein in modern biochemical assays. It forms nearly irreversible complexes (Kd ≈ 10-15 M) with the vitamin biotin and biotinylated molecules, which can be detected by a variety of methods.2-D streptavidin crystals were grown on biotinylated lipid monolayers by a method similar to that of Kubalek, et al., 1991. They were preserved in vitreous ice for electron cryomicroscopy. Electron images were recorded at 400 kV under flood beam illumination at a dose of 8 e-/Å2. Phases from four micrographs were merged.