CTF determination and correction for low dose tomographic tilt series

Xiong, Quanren; Morphew, Mary K.; Schwartz, Cindi L.; Hoenger, Andreas; Mastronarde, David N.

doi:10.1016/j.jsb.2009.08.016

Cited by 213 publications

(183 citation statements)

References 24 publications

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“…An input/output library for the MRC file format maintains the header information generated during collection throughout the processing. The main executable encompasses the following: drift correction of dose-fractionated data using dosefgpu_driftcorr (29) and the assembly of corrected sums into tilt series, automatic fiducial seed model generation by RAPTOR software (44), alignment and contrast transfer function correction of tilt series by IMOD software (45,46), and reconstruction of tilt series into tomograms by TOMO3D software (47). The flowchart is shown in SI Appendix, Fig.…”

Section: Methodsmentioning

confidence: 99%

Visualization of the type III secretion sorting platform of Shigella flexneri

Hu¹,

Morado²,

Margolin³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

204

300

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Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.nanomachine | injectisome | pathogen-host interaction | cryo-electron tomography | protein secretion

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Section: Methodsmentioning

confidence: 99%

Visualization of the type III secretion sorting platform of Shigella flexneri

Hu¹,

Morado²,

Margolin³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

204

300

View full text Add to dashboard Cite

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“…Each stack contains about eight images, which were first aligned using MotionCorr (66) and were assembled into the drift-corrected stacks. TOMOAUTO was used to facilitate the alignment in IMOD (63,67). All aligned tilt series were reconstructed into tomograms using TOMO3D (68).…”

Section: Methodsmentioning

confidence: 99%

Imaging the Motility and Chemotaxis Machineries in Helicobacter pylori by Cryo-Electron Tomography

et al. 2017

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Helicobacter pylori is a bacterial pathogen that can cause many gastrointestinal diseases, including ulcers and gastric cancer. A unique chemotaxis-mediated motility is critical for H. pylori to colonize in the human stomach and to establish chronic infection, but the underlying molecular mechanisms are not well understood. Here, we employ cryo-electron tomography (cryo-ET) to reveal detailed structures of the H. pylori cell envelope, including the sheathed flagella and chemotaxis arrays. Notably, H. pylori possesses a distinctive periplasmic cage-like structure with 18-fold symmetry. We propose that this structure forms a robust platform for recruiting 18 torque generators, which likely provide the higher torque needed for swimming in high-viscosity environments. We also reveal a series of key flagellar assembly intermediates, providing structural evidence that flagellar assembly is tightly coupled with the biogenesis of the membrane sheath. Finally, we determine the structure of putative chemotaxis arrays at the flagellar pole, which have implications for how the direction of flagellar rotation is regulated. Together, our pilot cryo-ET studies provide novel structural insights into the unipolar flagella of H. pylori and lay a foundation for a better understanding of the unique motility of this organism.IMPORTANCE Helicobacter pylori is a highly motile bacterial pathogen that colonizes approximately 50% of the world's population. H. pylori can move readily within the viscous mucosal layer of the stomach. It has become increasingly clear that its unique flagella-driven motility is essential for successful gastric colonization and pathogenesis. Here, we use advanced imaging techniques to visualize novel in situ structures with unprecedented detail in intact H. pylori cells. Remarkably, H. pylori possesses multiple unipolar flagella, which are driven by one of the largest flagellar motors found in bacteria. These large motors presumably provide the higher torque needed by the bacterial pathogens to navigate in the viscous environment of the human stomach.

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“…Theoretical contrast transfer function (CTF)-curves were fitted to averaged power spectra from 512 square pixel tiles generated from all images in a tilt series. CTF correction was done using the program "ctf phase flip" implemented in IMOD (35). Subvolumes with a size of 248 Å 3 were extracted from 2ϫ binned, CTF-corrected tomograms (pixel size, 2.07 Å) at the positions determined in the 8ϫ binned alignments.…”

Section: Methodsmentioning

confidence: 99%

The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly

Schur

Dick

Hagen

et al. 2015

J Virol

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The polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to study in vitro-assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of ϳ8 Å. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation. IMPORTANCERetroviruses first assemble into immature virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the virus into its infectious form. It is important to understand how Gag is arranged within immature retroviruses, in order to understand how virus assembly occurs, and how maturation takes place. We used the techniques cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in immature assembled Rous sarcoma virus Gag particles. We found that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the virus assembles and undergoes maturation.T he major structural component of retroviruses is the polyprotein Gag. The expression of Gag alone is sufficient to mediate the assembly and release of virus-like particles (VLPs). Gag proteins from all retroviruses contain an N-terminal membranebinding matrix (MA) domain, two capsid (CA) domains, and a nucleocapsid (NC) domain (1). These domains are structurally similar across retroviral genera but differ greatly in sequence. Outside these core do...

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CTF determination and correction for low dose tomographic tilt series

Cited by 213 publications

References 24 publications

Visualization of the type III secretion sorting platform of Shigella flexneri

Visualization of the type III secretion sorting platform of Shigella flexneri

Imaging the Motility and Chemotaxis Machineries in Helicobacter pylori by Cryo-Electron Tomography

The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly

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