Three-dimensional reconstruction of a freeze-dried and metal-shadowed bacterial surface layer

Three-dimensional reconstructions were performed from tilt series of a rotary-shadowed regular surface layer. Two series, representing the two surfaces of the HPI layer, were processed. The metal film stands out very clearly from the surrounding matter (protein, carbon) in the reconstructions and can, in good approximation, be regarded as reproducing the surface geometry of the protein layer. Thus, by combining information from both surfaces, it is possible to reconstruct profiles of the protein structure. Shadowing is advantageous in that it provides excellent reproducibility in specimen contrast, and the metal film is stable in the electron beam. Moreover, unlike negative staining, information is obtained from surfaces that are not in contact with the specimen support, and the technique is also applicable to freeze-dried material. (21 References)