Structural Analysis of Protein Complexes by Cryo-Electron Microscopy.

Area of Science:

• Structural biology
• Biophysics
• Molecular imaging

Background:

• Single particle cryo-electron microscopy (cryo-EM) has emerged as a powerful technique, rivaling X-ray crystallography for structural determination of bio-complexes.
• Advances in digital imaging and image processing algorithms have enabled near-atomic resolution (1.4–3 Å) for a wide range of biological macromolecules.
• While stable complexes like apoferritin are well-resolved, flexible and multi-component systems, such as bacterial secretion systems, present significant structural characterization challenges.

Approach:

• This review outlines the fundamental principles of sample preparation, digital data acquisition, and image analysis workflows essential for cryo-EM.
• It focuses on common methodologies applicable to both small and large biological complexes, aiming for high-resolution structure reconstruction and refinement.
• The practical application of these cryo-EM techniques is illustrated through the analysis of Type IV Secretion Systems.

Key Points:

• Cryo-EM routinely achieves resolutions better than 3 Å, with nearly 1200 structures deposited in the EMDB in 2022.
• The technique is particularly valuable for studying large, flexible, and non-crystallizable biological assemblies.
• Standardized protocols for data processing facilitate the determination of complex molecular architectures.

Conclusions:

• Cryo-EM is a versatile and increasingly indispensable tool for elucidating the structures of complex biological systems.
• The described workflow provides a framework for researchers to tackle challenging structural biology problems, including the analysis of dynamic protein machineries.
• Further application of these methods will continue to advance our understanding of molecular mechanisms in biology.