Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain...

Negative stain electron microscopy (NSEM) is a simple and cost effective method to analyze a wide variety of specimens, especially proteins. In traditional NSEM, the protein sample is applied to and supported by a continuous carbon film. Unfortunately, many proteins stick to the carbon film with a limited number of orientations. Because the restricted orientation limits the available views of the molecule, information about the three-dimensional structure of the molecule is likewise limited. The method presented here overcomes this limitation by using a carbon holey film combined with 1-octadecanol as a spreading agent. We demonstrate this method with solubilized envelope (Env) proteins from HIV, which typically show a restricted orientation on continuous carbon film, and show the following: •1-octadecanol added to negative stain aids the formation of a continuous sample-stain layer spanning the holes of a holey carbon film.•Samples negatively stained over holes show less restricted orientation, resulting in better single particle reconstructions.