Self-Perforated Hydrogel Nanomembranes Facilitate Structural Analysis of Proteins by Electron Cryo-Microscopy

We developed a method to improve specimen preparation for electron cryo-microscopy of membrane proteins. The method features a perforated hydrogel nanomembrane that stabilizes the thin film of aqueous buffer spanning the holes of holey carbon films, while at the same time preventing the depletion of protein molecules from these holes. The membrane is obtained by cross-linking of thiolated polyglycerol dendrimer films on gold, which self-perforate upon transfer to holey carbon substrates, forming a sub-micron-sized hydrogel network. The perforated nanomembrane improves the distribution of the protein molecules in the ice considerably. This facilitates data acquisition as demonstrated with two eukaryotic membrane protein complexes.