This article describes the transition of the Glass laboratory from X-ray crystallography to single-particle cryo-electron microscopy (cryo-EM) for structural studies of ATAD2B, a large AAA+ ATPase- and bromodomain-containing protein involved in chromatin regulation. It outlines practical strategies for protein expression, sample preparation, data processing and model building, offering a comprehensive workflow and troubleshooting guide for new cryo-EM users working with large, flexible macromolecular complexes.

Cryo-EM single-particle analysis has revolutionized structural biology by allowing high-resolution analysis of large molecular assemblies that are not amenable to crystallography or NMR. Combination of this technology with innovative sample-preparation protocols further expands the range of potential targets.

The most often used negative stains for single-particle analysis are based on the radioactive element uranium. Here, two stains based on tungsten or molybdenum are re-established as nonradioactive substitutes to allow the use of negative-staining electron microscopy by a broader user base in structural biology projects.

A near-atomic resolution map was obtained for lumazine synthase while benchmarking a new microscope. At this resolution, waters, ligands and hydrogens were visible. A detailed outline of the methods used is presented that can employed for any single-particle cryo-EM experiment.