Cryo-SiN – An Alternative Substrate to Visualize Active Viral Assemblies

Silicon nitride (SiN) membranes that are merely nanometers-thick have been introduced as new support films for electron microscopy (EM) applications. The use of these thin membranes for high-resolution imaging is widely attractive due to their consistent manufacture, flatness, and durability. Here, we utilized SiN support films to actively monitor key steps in rotavirus RNA synthesis at the nanoscale. Rotaviruses are gastrointestinal pathogens that infect pediatric populations and pose a major health problem in developing countries. We demonstrate, for the first time, that cryo-EM specimens of these viral assemblies may be prepared using SiN membranes, that we have termed “cryo-SiN”. Cryo-SiN microchips containing inactive and actively transcribing rotavirus complexes were vitrified using gentle blot conditions to yield reproducibly thin ice-embedded specimens. EM imaging results indicate that cryo-SiN samples exhibited greater visual contrast than specimens prepared on conventional support films under the same conditions. The improved contrast of the cryo-SiN samples permitted us to calculate 3D structures of rotavirus complexes using a fraction of the images required for conventionally prepared specimens. We anticipate the use of cryo-SiN microchip will enable us to better investigate critical viral life-cycle processes at the molecular level under near-native conditions.