Setup and Troubleshooting of Volta Phase Plate Cryo-EM Data Collection.

Abstract

Cryo\xa0electron microscopy (cryo-EM) has become a method of choice in structural biology to analyze isolated complexes and cellular structures. This implies adequate imaging of the specimen and advanced image-processing methods to obtain high-resolution 3D reconstructions. The use of a Volta phase plate in cryo-EM drastically increases the image contrast while being able to record images at high acceleration voltage and close to focus, i.e., at conditions where high-resolution information is best preserved. During image processing, higher contrast images can be aligned and classified better than lower quality ones resulting in increased data quality and the need for less data. Here, we give step-by-step guidelines on how to set up high-quality VPP cryo-EM single particle data collections, as exemplified by human ribosome data acquired during a one-day data collection session. Further, we describe specific technical details in image processing that differ from conventional single particle cryo-EM data analysis.