Breaking barriers: transitioning from X-ray crystallography to cryo-EM for structural studies

Cryo-electron microscopy (cryo-EM) has transformed structural biology by enabling near-atomic resolution of large macromolecular complexes without the need for crystallization. Here, we describe our laboratory's transition from X-ray crystallography to single-particle cryo-EM to investigate the ATPase family AAA+ domain-containing protein 2B (ATAD2B), a chromatin regulator implicated in epigenetic signaling. We outline the challenges encountered during protein expression, purification and sample preparation, including co-purification of the chaperonin GroEL, and the strategies employed to overcome these obstacles. Our workflow highlights critical steps in sample optimization, grid vitrification and data processing using CryoSPARC , cisTEM and Topaz , as well as computational requirements for high-resolution reconstructions. We also discuss model-building, refinement and validation approaches, emphasizing best practices for new cryo-EM users. This work provides practical insights for structural biologists adopting cryo-EM, particularly for large, flexible protein complexes, and underscores the importance of integrated approaches combining biochemical, computational and imaging strategies.