THE NEGATIVE STAINING CARBON-FILM PROCEDURE - TECHNICAL CONSIDERATIONS AND A SURVEY OF MACROMOLECULAR APPLICATIONS

The development and initial virological applications of the negative staining-carbon film (NS-CF) procedure are described and followed by a survey of some recent macromolecular applications of the technique. Electron microscopical data are shown which indicate the capability of the technique for the production of two-dimensional (2-D) crystals from several different soluble proteins (human erythrocyte catalase, human erythrocyte, Xenopus and Thermoplasma acidophilum cylindrin/multicatalytic proteinase, yeast glutamine synthetase and keyhole limpet haemocyanin). Instances in which ordered two-dimensional close-packing of molecules is produced, rather than true two-dimensional crystallization, are also indicated. In addition, data obtained from the application of the NS-CF procedure to a number of fibrillar proteins are briefly presented. Examples include bovine lens vimentin, thrombin-cleaved vimentin and bacterially expressed Xenopus lamin-A. These elongated proteins form higher order filamentous structures, networks and, in the case of lamin-A, large paracrystalline structures. A general discussion of the achievements and future potential of the NS-CF procedure is presented.