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

Abstract

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions. 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.

In Situ Molecular Architecture of the Salmonella Type III Secretion Machine

“…S1). Our high-throughput cryo-ET pipeline effectively integrates dose-fractionation in a direct detector device with specific software, allowing massive data collection, drift correction, fiducial model generation, alignment, contrast transfer function (CTF) correction, and reconstruction of several thousands of frozen-hydrated minicells at high magnification (Morado et al, 2016). A typical three-dimensional (3D) reconstruction of a Salmonella minicell revealed multiple injectisomes embedded in the cell envelope (Fig.…”

“…Each stack contains ~8 images. To analyze over 60 TB raw data from the microscope and the direct detection device, we used Tomoauto (Morado et al, 2016) to facilitate image processing: drift correction of dose-fractionated data using Motioncorr (Li et al, 2013) assembly of corrected sums into tilt series, automatic fiducial seed model generation, alignment, defocus estimation, and contrast transfer function correction of tilt series using IMOD (Kremer et al, 1996), and weighted back projection (WBP) reconstruction of tilt series into tomograms using Tomo3D (Agulleiro and Fernandez, 2015). Each tomographic reconstruction is 3,710 × 3,838 × 1,800 voxels and ~100Gb in size.…”

Ultrastructural analysis of bacteriophage Φ29 during infection of Bacillus subtilis

“…Using SerialEM (Mastronarde, 2005), approximately 350 low-dose, single-axis tilt series were collected at −6 to −8 μm defocus with a cumulative dose of ~60 e − /Å 2 distributed over 41 images and covering an angular range of −60° to +60°, with angular increments of 3°. Tilt series were drift corrected (Li et al, 2013), aligned using IMOD (Kremer et al, 1996) through Tomoauto (Morado et al, 2016), and reconstructed using Tomo3D (Agulleiro and Fernandez, 2015). In total, 308 tomographic reconstructions were generated by using Weighted Back Projection (WBP) and used for further processing (Table S1).…”

The flagellar motor ofVibrio alginolyticusundergoes major structural remodeling during rotational switching

“…For every single tilt series collection, the dose-fractionated mode was used to generate 8 to 10 frames per projection image. Collected dose-fractionated data were first subjected to the motion correction program to generate drift-corrected stack files (Li et al, 2013;Morado et al, 2016). The stack files were aligned using gold fiducial markers and volumes reconstructed using IMOD and Tomo3d, respectively (Agulleiro and Fernandez, 2015;Kremer et al, 1996).…”