Preparation of RNA Polymerase Complexes for Their Analysis by Single-Particle Cryo-Electron Microscopy

Abstract

AbstractRecent technological progress revealed new prospects of high-resolution structure determination of macromolecular complexes using cryo-electron microscopy (cryo-EM). In the field of RNA polymerase (Pol) I research, a number of cryo-EM studies contributed to understanding the highly specialized mechanisms underlying the transcription of ribosomal RNA genes. Despite a broad applicability of the cryo-EM method itself, preparation of samples for high-resolution data collection can be challenging. Here, we describe strategies for the purification and stabilization of Pol I complexes, exemplarily considering advantages and disadvantages of the methodology. We further provide an easy-to-implement protocol for the coating of EM-grids with self-made carbon support films. In sum, we present an efficient workflow for cryo-grid preparation and optimization, including early stage cryo-EM screening that can be adapted to a wide range of soluble samples for high-resolution structure determination.