title: Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography.
abstract: 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.
authors: Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston.
authors: Dustin R
authors: DR
authors: Morado
authors: Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston.
authors: Bo
authors: B
authors: Hu
authors: Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston; Jun.Liu.1@uth.tmc.edu.
authors: Jun
authors: J
authors: Liu
mesh_terms: D001331:Automation / Q000379:methods; D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D007091:Image Processing, Computer-Assisted / Q000379:methods; D021621:Imaging, Three-Dimensional / Q000379:methods; D046911:Macromolecular Substances; D012763:Shigella flexneri / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D012984:Software; D000069296:Type III Secretion Systems / Q000032:analysis / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D000069296:Type III Secretion Systems