Obtaining high-resolution cryo-EM structures using a common LaB6, 120-keV electron microscope equipped with a sub 200-keV optimised direct electron detector.

Abstract

Cryo-electron microscopy (Cryo-EM) single particle analysis (SPA) has become a major structural biology technique in recent years. High-resolution cryo-EM typically requires higher voltage cryo-TEMs with coherent FEG sources, stable columns, autoloader systems and direct electron detectors. These setups are specialised for Cryo-EM work and are expensive to establish and maintain. More recently the concept of using 100-keV cryo-TEMs has been introduced as a way to make cryo-EM more affordable and hence accessible to a larger group of researchers. So far, the implementation of these 100-keV cryo-TEMs have relied on specialised microscopes with FEG sources as well as more stable optics than usually present on the common 120-keV TEMs. We here explored whether a standard 120-keV TEM, commonly available at many laboratories worldwide, can be upgraded with a direct electron detector and its suitability for high-resolution cryo-EM using a standard side entry cryo-holder. Using this imaging configuration, we were successful in achieving a 2.65[A] reconstruction for standard apoferritin. We were also able to resolve a more challenging small 64kDa protein haemoglobin to 4.33[A]. Furthermore, we were able to solve an asymmetric 153 kDa membrane protein GPCR (M4 muscarinic acid receptor) to a resolution of 4.4[A]. Importantly, all these results were achieved using a standard automated data collection routine implemented through SerialEM, making it feasible to collect large cryo-EM data sets with a side entry cryo-holder. These results showcase a potentially widely accessible solution to obtaining interpretable cryo-EM structures. Furthermore, we envisage that this imaging configuration gives an option for many EM facilities and laboratories to set up a high-quality cryo-EM SPA sample screening capability without the need to procure costly specialised Cryo-TEMs. This could help to considerably lower the economic entry barrier for cryo-EM SPA and contribute to the "democratisation" of cryo-EM.