Preparing Frozen-Hydrated Protein-Nucleic Acid Assemblies for High-Resolution Cryo-EM Imaging.

High-resolution image acquisition and structure determination by cryo-electron microscopy is becoming increasingly streamlined. Preparing electron-microscopy grids of suitable quality remains, however, a critical bottleneck. Strategies to achieve particle monodispersity, optimal sample concentration and suitable ice thickness can vary from specimen to specimen. In this book chapter we describe our protocols for negative-stain grid and cryo-grid preparation, which we apply to studying protein-nucleic acid complexes.