Preparing frozen-hydrated protein-nucleic acid assemblies for high-resolution cryo-EM imaging

Abstract

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.