Quantitative evaluation of heavy metal decoration on protein molecules: contrast, specificity and resolution

Replication by heavy metal decoration is a technique capable of portraying topochemical properties of biological surfaces. The technique has already been applied to study molecular symmetries and crystal packing of large, oligomeric protein complexes. A quantitative approach was used to analyse the decoration pattern of various metals on freeze-etched protein surfaces utilizing modified image analysis techniques. The normalized metal distribution was derived from optical densities of the electron micrographs and of their correlation average. Averaging of the centres of mass of the metal clusters, which reveals the spatial distribution of the clusters, led to an improved lateral resolution and to a reduced influence of the surface relief on the decoration pattern. Differing size distributions of the clusters proved to be an additional parameter being characteristic for specific surface sites, as demonstrated by separately averaging of clusters of different sizes. Making use of these procedures, decoration efficacies of different surface sites on a molecule and also decoration patterns obtained by modifying either the proteins surface or the technique were compared quantitatively, allowing definite discrimination between maxima located at surface sites of different chemical nature.