Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Area of Science:

• Structural Biology
• Microscopy Techniques
• Biophysics

Background:

• Cryo-electron tomography (Cryo-ET) enables molecular-level 3-D visualization of biological samples in their native state.
• Low signal-to-noise ratios in raw cryo-tomograms necessitate advanced image processing techniques like sub-tomogram averaging.
• Acquiring large datasets for Cryo-ET is often a bottleneck in structural analysis.

Purpose of the Study:

• To present a high-throughput cryo-electron tomography (Cryo-ET) protocol designed to overcome data acquisition and processing challenges.
• To demonstrate the application of this protocol for visualizing complex biological systems at high resolution.
• To provide a broadly applicable method for researchers utilizing Cryo-ET.

Main Methods:

• Implementation of a high-throughput Cryo-ET workflow using a 300kV cryo-electron microscope and a direct detection device (DDD) camera.
• Development and application of an in-house, semi-automated image-processing pipeline software library called tomoauto.
• Collection of large tilt-series datasets crucial for averaging and classification of macromolecular complexes.

Main Results:

• Successfully visualized the intact type III secretion system (T3SS) in Shigella flexneri minicells using the developed protocol.
• The protocol significantly enhances throughput for Cryo-ET data acquisition and processing.
• Demonstrated the effectiveness of the semi-automated software pipeline in managing large datasets.

Conclusions:

• The described high-throughput Cryo-ET protocol offers an efficient solution for molecular imaging challenges.
• This method facilitates detailed structural analysis of macromolecular complexes, including the T3SS.
• The protocol is adaptable for diverse research projects requiring advanced Cryo-ET analysis.