Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation

作者： Trinity Cookis1, Paul Sauer2,3, Simon Poepsel4,5, Bong-Gyoon Han6, Dominik A. Herbst1,2,6, Robert Glaeser6, Eva Nogales1,2,3,6 1Department of Molecular and Cell Biology,University of California, Berkeley, 2California Institute for Quantitative Biology (QB3),University of California, Berkeley, 3Howard Hughes Medical Institute,University of California, Berkeley, 4Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital,University of Cologne, 5Cologne Excellence Cluster for Cellular Stress Responses in Ageing-Associated Diseases (CECAD),University of Cologne, 6Molecular Biophysics and Integrative Bio-Imaging Division,Lawrence Berkeley National Laboratory

简介：

Overview

This article presents a detailed protocol for fabricating streptavidin affinity grids, which are essential for structural studies of challenging macromolecular samples using cryo-electron microscopy (cryo-EM). The method addresses common issues in sample preparation, such as protein denaturation and orientation biases.

Key Study Components

Area of Science

• Cryo-electron microscopy
• Structural biology
• Macromolecular analysis

Background

• Recent advancements in cryo-EM technology have improved image quality.
• Sample preparation is a significant bottleneck in cryo-EM workflows.
• Proteins can denature or orient preferentially during sample preparation.
• Streptavidin-biotin interactions can stabilize samples and mitigate these issues.

Purpose of Study

• To provide a reliable protocol for preparing streptavidin affinity grids.
• To enhance the quality of cryo-EM images by reducing sample denaturation.
• To address preferential orientation problems in cryo-EM samples.

Methods Used

• Cleaning and preparing glass syringes and grids with ethanol and chloroform.
• Creating lipid monolayers to stabilize biotinylated samples.
• Incubating grids with streptavidin and rinsing to remove unbound proteins.
• Using carbon evaporation to prepare grids for imaging.

Main Results

• The protocol successfully produced grids that maintained sample integrity.
• Micrographs showed a continuous grid pattern, indicating effective lattice formation.
• Fast Fourier transform analysis confirmed the removal of streptavidin signals.
• The method allows for the use of low sample amounts without compromising quality.

Conclusions

• The streptavidin affinity grid protocol is effective for cryo-EM studies.
• This approach can significantly improve the analysis of challenging macromolecular samples.
• Future applications may further enhance structural biology research.

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