Electron microscopic observations on protein crystallization: adsorption layers, aggregates and crystal defects

Transmission electron microscopy of freeze-etched and heavy-metal-decorated large protein complexes is capable of portraying their molecular symmetrie s and orientations. The technique in combination with image analysis has be en applied to study the rotational order of individual lumazine synthase mo lecules either in crystals or adsorbed on solid substrates. On crystal surf aces rotational disorder was mainly observed for the molecules at and in th e vicinity of relief perturbations. Molecules along surface steps and in th e vicinity of vacancies showed no noticeable deviation from their translati onal and rotational order dictated by the lattice. Adsorption of lumazine s ynthase on mica led to layers with different degrees of coverage. Single, i solated molecules showed a slight preferential orientation with a hydrophob ic region of their surface in contact with the substrate. In continuous, mo nomolecular adsorption layers microclusters have been observed in which the neighboring molecules were oriented in the same way with respect to the su bstrate. These microclusters may act as nuclei for the second layer which r evealed a perfect crystalline order as do the consecutive layers. Thus, the multilayer structures grown on mica present real three-dimensional crystal s except for the first layer contacting the substrate. (C) 2000 Elsevier Sc ience B.V. All rights reserved.