What Could Go Wrong? A Practical Guide to Single-Particle Cryo-EM: From Biochemistry to Atomic Models

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Cryo-electron microscopy (cryo-EM) is rapidly advancing, enabling high-resolution protein structure determination.
• The technique allows visualization of molecular structures without the need for crystallization.
• Increased accessibility to cryo-EM necessitates understanding its common pitfalls.

Purpose of the Study:

• To identify and address common misconceptions in initiating cryo-electron microscopy projects.
• To outline practical solutions for technical hurdles encountered in cryo-EM structure determination.
• To provide insights into the future trajectory of cryo-EM applications.

Main Methods:

• Review and analysis of common challenges in cryo-electron microscopy workflows.
• Identification of misconceptions hindering successful high-resolution structure determination.
• Discussion of practical strategies and solutions for experimental design and data processing.

Main Results:

• Detailed description of frequent technical pitfalls in sample preparation, data collection, and image processing.
• Clarification of common misunderstandings regarding instrument parameters and data quality assessment.
• Presentation of actionable solutions to overcome these challenges for improved structural outcomes.

Conclusions:

• Successful high-resolution cryo-EM structure determination requires careful consideration of numerous project-specific factors.
• Addressing common misconceptions and technical hurdles is crucial for efficient and reliable results.
• The field of cryo-EM is poised for continued innovation and broader application in structural biology.