UTILITY OF BUTVAR SUPPORT FILM AND METHYLAMINE TUNGSTATE STAIN IN 3-DIMENSIONAL ELECTRON-MICROSCOPY - AGREEMENT BETWEEN STAIN AND FROZEN-HYDRATED RECONSTRUCTIONS

A random conical tilt reconstruction of negatively stained Saccharomyc es cerevisiae fatty acid synthase was used as a model to compute a thr ee-dimensional reconstruction from untilted stain specimens of the mol ecules in multiple orientations using a three-dimensional projection a lignment method. The resulting structure (24 Angstrom resolution) has a more uniform resolution than the initial structure and the handednes s revealed in the random conical tilt method is preserved, In a simila r approach, this model was used to compute a 21-Angstrom-resolution fr ozen-hydrated structure from untilted specimens of the molecules in mu ltiple orientations. Even though the reconstructions are in close agre e ment, the stain structure appears to enhance the protein density ass ociated with less robust features. These procedures significantly redu ce the time and effort required to obtain a three-dimensional reconstr uction from frozen-hydrated data with a resolution that is comparable to the best obtained by more laborious methods. The agreement between the stain and frozen-hydrated reconstructions affords convincing evide nce concerning the validity of the structure and the information affor ded by the two reconstructions significantly enhances the structural a nalysis of the molecule. (C) 1997 Academic Press.