Tomo3D 2.0 – Exploitation of Advanced Vector eXtensions (AVX) for 3D reconstruction

Agulleiro, J.I.; Fernández, José‐Jesús

doi:10.1016/j.jsb.2014.11.009

Cited by 169 publications

(116 citation statements)

References 18 publications

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“…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.…”

Section: Star* Methodsmentioning

confidence: 99%

In Situ Molecular Architecture of the Salmonella Type III Secretion Machine

Hu¹,

Lara‐Tejero

Kong

et al. 2017

Cell

190

323

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Type III protein secretion systems have specifically evolved to deliver bacterially encoded proteins into target eukaryotic cells. The core elements of this multi-protein machine are the envelope-associated needle complex, the inner membrane export apparatus, and a large cytoplasmic sorting platform. Here we report a high-resolution in situ structure of the Salmonella Typhimurium type III secretion machine obtained by high-throughput cryo-electron tomography and sub-tomogram averaging. Through molecular modeling and comparative analysis of machines assembled with protein-tagged components or from different deletion mutants we determined the molecular architecture of the secretion machine in situ and localized its structural components. We also show that docking of the sorting platform results in significant conformational changes in the needle complex to provide the symmetry adaptation required for the assembly of the entire secretion machine. These studies provide major insight into the structure and assembly of a broadly distributed protein secretion machine.

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Section: Star* Methodsmentioning

confidence: 99%

In Situ Molecular Architecture of the Salmonella Type III Secretion Machine

Hu¹,

Lara‐Tejero

Kong

et al. 2017

Cell

190

323

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“…Tomoauto has been designed so that the user may use IMOD 13 or tomo3d 26 to generate the final reconstruction. We currently use tomo3d to take advantage of several features in modern multi-core computer processing units (CPUs) to greatly reduce the reconstruction time.…”

Section: Representative Resultsmentioning

confidence: 99%

“…Alternatively users have the option in tomoauto to use RAPTOR 24 (included in IMOD) for automated fiducial model generation, CTFFIND4 25 (http://grigoriefflab.janelia.org/ctf) to determine the CTF, and tomo3d 26 (https://sites.google.com/site/3demimageprocessing/tomo3d) for reconstruction or in any combination of software packages by configuration. This configuration as well as the parameters available in each package is handled by a global configuration file that can be edited to suit the values most commonly used in a lab while local configuration files can also be created to detail parameters used for a specific specimen, collection set or individual tilt-series.…”

Section: High-throughput Automated Tilt-series Processing and Recomentioning

confidence: 99%

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

Morado

2016

JoVE

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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.

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“…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).…”

Section: Methodsmentioning

confidence: 99%

Ultrastructural analysis of bacteriophage Φ29 during infection of Bacillus subtilis

Farley¹,

Tu²,

Kearns

et al. 2017

Journal of Structural Biology

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Recent advances in cryo-electron tomography (cryo-ET) have allowed direct visualization of the initial interactions between bacteriophages and their hosts. Previous studies focused on phage infection in Gram-negative bacteria but it is of particular interest how phages penetrate the thick, highly cross-linked Gram-positive cell wall. Here we detail structural intermediates of phage Φ29 during infection of Bacillus subtilis. Use of a minicell-producing strain facilitated in situ tomographic reconstructions of infecting phage particles. Φ29 initially contacts the cell wall at an angle through a subset of the twelve appendages, which are attached to the collar at the head proximal portion of the tail knob. The appendages are flexible and switch between extended and downward conformations during this stage of reversible adsorption; appendages enzymatically hydrolyze wall teichoic acids to bring the phage closer to the cell. A cell wall-degrading enzyme at the distal tip of the tail knob locally digests peptidoglycan, facilitating penetration of the tail further into the cell wall, and the phage particle reorients so that the tail becomes perpendicular to the cell surface. All twelve appendages attain the same “down” conformation during this stage of adsorption. Once the tail has become totally embedded in the cell wall, the tip can fuse with the cytoplasmic membrane. The membrane bulges out, presumably to facilitate genome ejection into the cytoplasm, and the deformation remains after complete ejection. This study provides the first visualization of the structural changes occurring in a phage particle during adsorption and genome transfer into a Gram-positive bacterium.

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Tomo3D 2.0 – Exploitation of Advanced Vector eXtensions (AVX) for 3D reconstruction

Cited by 169 publications

References 18 publications

In Situ Molecular Architecture of the Salmonella Type III Secretion Machine

In Situ Molecular Architecture of the Salmonella Type III Secretion Machine

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

Ultrastructural analysis of bacteriophage Φ29 during infection of Bacillus subtilis

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