Macromolecular 3D SEM reconstruction strategies: Signal to noise ratio and resolution

Three-dimensional scanning electron microscopy generates quantitative volumetric structural data from SEM images of macromolecules. This technique provides a quick and easy way to define the quaternary structure and handedness of protein complexes. Here, we apply a variety of preparation and imaging methods to filamentous actin in order to explore the relationship between resolution, signal-to-noise ratio, structural preservation and dataset size. This information can be used to define successful imaging strategies for different applications. - Highlights: • F-actin SEM datasets were collected using 8 different preparation/ imaging techniques. • Datasets were reconstructed by back projection and compared/analyzed • 3DSEM actin reconstructions can be produced with <100 views of the asymmetric unit. • Negatively stained macromolecules can be reconstructed by 3DSEM to ∼3 nm resolution