Structural preservation and absolute contrast of catalase crystal sections prepared with tannic acid

Abstract

Three methods of obtaining 20 Å resolution in sectioned protein crystals have recently been described. They include tannic acid fixation, low temperature embedding and grid sectioning. To be useful for 3-dimensional reconstruction thin sections must possess suitable resolution, structural fidelity and a known contrast. Tannic acid fixation appears to satisfy the above criteria based on studies of crystals ofPseudomonascytochrome oxidase, orthorhombic beef liver catalase and beef heart F1-ATPase. In order to develop methods with general applicability, we have concentrated our efforts on a trigonal modification of catalase which routinely demonstrated a resolution of 40 Å. The catalase system is particularly useful since a comparison with the structure recently solved with x-rays will permit evaluation of the accuracy of 3-D reconstructions of sectioned crystals.Initially, we re-evaluated the packing of trigonal catalase crystals studied by Longley. Images of the (001) plane are of particular interest since they give a projection down the 31-screw axis in space group P3121. Images obtained by the method of Longley or by tannic acid fixation are negatively contrasted since control experiments with orthorhombic catalase plates yield negatively stained specimens with conditions used for the larger trigonal crystals.