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

• Published in: Journal of visualized experiments no. 107; p. e53608
• Main Authors: Morado, Dustin R., Hu, Bo, Liu, Jun
• Format: Journal Article
• Language: English
• Published: United States MyJove Corporation 30.01.2016
• Subjects: automation; Automation - methods; Bioengineering; cameras; computer software; Cryoelectron Microscopy - methods; data collection; Electron Microscope Tomography - methods; freezing; High-Throughput Screening Assays - methods; ice; image analysis; Image Processing, Computer-Assisted - methods; Imaging, Three-Dimensional - methods; Macromolecular Substances; mutation; Shigella flexneri; Shigella flexneri - chemistry; Shigella flexneri - cytology; Shigella flexneri - metabolism; signal-to-noise ratio; Software; tomography; type III secretion system; Type III Secretion Systems - analysis; Type III Secretion Systems - metabolism
• ISSN: 1940-087X; 1940-087X
• DOI: 10.3791/53608

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.