Cryo-EM single-particle analysis expanding towards increasingly native samples.

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Cryo-electron microscopy (cryo-EM) has revolutionized structural biology.
• Single-particle analysis (SPA) typically requires purified proteins.
• Cryo-electron tomography (cryo-ET) visualizes structures within cells but has resolution limits.

Purpose of the Study:

• To highlight advancements in cryo-EM single-particle analysis (SPA).
• To showcase SPA's expanding applicability to native and complex biological samples.
• To demonstrate SPA as a tool for de novo protein identification.

Main Methods:

• Review of recent studies utilizing cryo-EM SPA.
• Focus on tailored sample preparation strategies for complex assemblies.
• Comparison with cryo-focused ion beam (FIB) milling and cryo-electron tomography (cryo-ET).

Main Results:

• SPA is increasingly suitable for native and complex macromolecular assemblies.
• Tailored preparation enables SPA on previously challenging samples.
• SPA can now be used for de novo protein identification and complex stoichiometry.

Conclusions:

• Cryo-EM SPA is evolving beyond purified samples.
• Advanced sample preparation is key to analyzing native biological systems.
• SPA is a powerful tool for discovering protein complexes in their cellular context.