Utility of Butvar Support Film and Methylamine Tungstate Stain in Three-Dimensional Electron Microscopy: Agreement between Stain and Frozen-Hydrated Reconstructions

A random conical tilt reconstruction of negatively stainedSaccharomyces cerevisiaefatty acid synthase was used as a model to compute a three-dimensional reconstruction from untilted stain specimens of the molecules in multiple orientations using a three-dimensional projection alignment method. The resulting structure (24 Å resolution) has a more uniform resolution than the initial structure and the handedness revealed in the random conical tilt method is preserved. In a similar approach, this model was used to compute a 21-Å-resolution frozen-hydrated structure from untilted specimens of the molecules in multiple orientations. Even though the reconstructions are in close agreement, the stain structure appears to enhance the protein density associated with less robust features. These procedures significantly reduce the time and effort required to obtain a three-dimensional reconstruction 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 evidence concerning the validity of the structure and the information afforded by the two reconstructions significantly enhances the structural analysis of the molecule.