Know your detergents: A case study on detergent background in negative stain electron microscopy.

Area of Science:

• Structural biology
• Biophysical techniques
• Macromolecular complex analysis

Background:

• Electron cryo-microscopy (cryo-EM) provides near-atomic resolution 3D structures but requires highly optimized samples.
• Negative stain EM is a rapid, cost-effective preliminary method for sample quality control, crucial for cryo-EM and X-ray crystallography.
• Interpreting negative stain EM data requires careful consideration of potential artifacts, such as background from detergent micelles.

Purpose of the Study:

• To investigate and highlight the common artifact of background signal from detergent micelles in negative stain EM.
• To demonstrate how detergent micelles can be misinterpreted as protein samples, affecting sample quality assessment.
• To provide a template for evaluating negative stain EM images, particularly for small membrane proteins.

Main Methods:

• Case study analysis of commonly used detergents in negative stain EM.
• Microscopy evaluation of detergent behavior and background signal generation.
• Comparison of background signal across different detergents and concentrations relative to critical micelle concentration (CMC).

Main Results:

• Most detergents produce significant background signal in negative stain EM, even at concentrations below their nominal CMC.
• This background signal can be easily confused with actual detergent-embedded protein samples.
• Unawareness of this artifact can lead to incorrect assessments of sample quality and homogeneity.

Conclusions:

• Detergent-induced background is a prevalent issue in negative stain EM that can compromise data interpretation.
• Careful evaluation of negative stain EM images is essential to avoid misinterpreting detergent artifacts as protein signals.
• This study serves as a guide for researchers to identify and mitigate background artifacts in their EM sample quality control processes.