100 keV電子顕微鏡を用いた構造生物学のための統合ワークフロー

Cryo-EM has revolutionized structural biology, especially for flexible and heterogeneous samples, although access to high end microscopes that enable these studies remains a bottleneck. While 300 keV microscopes have been the go-to for high-resolution structural determination, they are expensive and restricted to institutional and national facilities needing specialized expertise, with access falling far short of the demand. Here, we present the user-managed operation of a cheaper 100 keV electron microscope within a structural biology laboratory enabling close integration with protein production, biochemical and biophysical studies. We provide details and considerations for the installation of the microscope, its day-to-day maintenance, and operations. Using virus surface glycoproteins as case studies, we illustrate the workflow from grid screening, data collection, and data processing, and provide examples of data quality. This user-administered setup provides a training platform for researchers at all levels, with beginners in cryo-EM achieving proficiency to independently operate the microscope within a month of regular use and training. We have demonstrated routine high-quality low-resolution reconstructions using a Ceta CMOS camera and high-resolution reconstructions enabling building of atomic models using a Falcon C direct detector. While there are several examples of facilities that manage cryo-EM and individual laboratories leveraging cryo-EM, we provide here the first demonstration of a modern group independently doing both successfully, something that has been talked about frequently but rarely seen.