Determination of the ribosome structure to a resolution of 2.5 Å by single-particle cryo-EM

With the advance of new instruments and algorithms, and the accumulation of experience over decades, single‐particle cryo‐EM has become a pivotal part of structural biology. Recently, we determined the structure of a eukaryotic ribosome at 2.5 Å for the large subunit. The ribosome was derived from Trypanosoma cruzi, the protozoan pathogen of Chagas disease. The high‐resolution density map allowed us to discern a large number of unprecedented details including rRNA modifications, water molecules, and ions such as Mg2+ and Zn2+. In this paper, we focus on the procedures for data collection, image processing, and modeling, with particular emphasis on factors that contributed to the attainment of high resolution. The methods described here are readily applicable to other macromolecules for high‐resolution reconstruction by single‐particle cryo‐EM.