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

Area of Science:

• Structural Biology
• Biochemistry
• Molecular Biology

Background:

• Cryo-electron microscopy (cryo-EM) enables near-atomic resolution of large macromolecular complexes without crystallization.
• The ATPase family AAA+ domain-containing protein 2B (ATAD2B) is a chromatin regulator involved in epigenetic signaling.

Purpose of the Study:

• To describe the transition from X-ray crystallography to single-particle cryo-EM for studying ATAD2B.
• To outline challenges and strategies in protein expression, purification, and sample preparation for cryo-EM.
• To provide practical insights for structural biologists adopting cryo-EM for large, flexible protein complexes.

Main Methods:

• Single-particle cryo-electron microscopy (cryo-EM) workflow.
• Protein expression and purification strategies.
• CryoSPARC, cisTEM, and Topaz for data processing.
• Model-building, refinement, and validation approaches.

Main Results:

• Successful application of cryo-EM to investigate ATAD2B.
• Overcoming challenges such as GroEL co-purification.
• Achieving high-resolution reconstructions through optimized sample preparation and data processing.

Conclusions:

• Cryo-EM is a powerful tool for structural biology, complementing traditional methods.
• Integrated biochemical, computational, and imaging strategies are crucial for success.
• This work offers valuable guidance for researchers new to cryo-EM, especially for challenging protein targets.