scholarly journals Fine details in complex environments: the power of cryo-electron tomography

2018 ◽  
Vol 46 (4) ◽  
pp. 807-816 ◽  
Author(s):  
Joshua Hutchings ◽  
Giulia Zanetti
Keyword(s):  
Structural Information ◽  
Electron Tomography ◽  
Molecular Structures ◽  
Complex Environments ◽  
Single Particle Reconstruction ◽  
Wide Range ◽  
Cryo Electron Tomography ◽  
Recent Developments

Cryo-electron tomography (CET) is uniquely suited to obtain structural information from a wide range of biological scales, integrating and bridging knowledge from molecules to cells. In particular, CET can be used to visualise molecular structures in their native environment. Depending on the experiment, a varying degree of resolutions can be achieved, with the first near-atomic molecular structures becoming recently available. The power of CET has increased significantly in the last 5 years, in parallel with improvements in cryo-EM hardware and software that have also benefited single-particle reconstruction techniques. In this review, we cover the typical CET pipeline, starting from sample preparation, to data collection and processing, and highlight in particular the recent developments that support structural biology in situ. We provide some examples that highlight the importance of structure determination of molecules embedded within their native environment, and propose future directions to improve CET performance and accessibility.

scholarly journals Multiscale Electron Microscopy for the Study of Viral Replication Organelles

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 197
Author(s):  
Georg Wolff ◽  
Montserrat Bárcena
Keyword(s):  
Electron Microscopy ◽  
Viral Replication ◽  
Electron Tomography ◽  
Viral Rna ◽  
Human Pathogens ◽  
Wide Range ◽  
Recent Developments ◽  
Versatile Technique ◽  
Positive Strand Rna

During infection with positive-strand RNA viruses, viral RNA synthesis associates with modified intracellular membranes that form unique and captivating structures in the cytoplasm of the infected cell. These viral replication organelles (ROs) play a key role in the replicative cycle of important human pathogens like coronaviruses, enteroviruses, or flaviviruses. From their discovery to date, progress in our understanding of viral ROs has closely followed new developments in electron microscopy (EM). This review gives a chronological account of this progress and an introduction to the different EM techniques that enabled it. With an ample repertoire of imaging modalities, EM is nowadays a versatile technique that provides structural and functional information at a wide range of scales. Together with well-established approaches like electron tomography or labeling methods, we examine more recent developments, such as volume scanning electron microscopy (SEM) and in situ cryotomography, which are only beginning to be applied to the study of viral ROs. We also highlight the first cryotomography analyses of viral ROs, which have led to the discovery of macromolecular complexes that may serve as RO channels that control the export of newly-made viral RNA. These studies are key first steps towards elucidating the macromolecular complexity of viral ROs.

scholarly journals Cryo-electron tomography: The challenge of doing structural biology in situ

2013 ◽  
Vol 202 (3) ◽  
pp. 407-419 ◽  
Author(s):  
Vladan Lučić ◽  
Alexander Rigort ◽  
Wolfgang Baumeister
Keyword(s):  
Cell Biology ◽  
Structural Information ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Molecular Structures ◽  
New Techniques ◽  
Preparation Methods ◽  
Technical Advances ◽  
Macromolecular Organization

Electron microscopy played a key role in establishing cell biology as a discipline, by producing fundamental insights into cellular organization and ultrastructure. Many seminal discoveries were made possible by the development of new sample preparation methods and imaging modalities. Recent technical advances include sample vitrification that faithfully preserves molecular structures, three-dimensional imaging by electron tomography, and improved image-processing methods. These new techniques have enabled the extraction of high fidelity structural information and are beginning to reveal the macromolecular organization of unperturbed cellular environments.

scholarly journals Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

2022 ◽  
Author(s):  
Jie E Yang ◽  
Matthew R Larson ◽  
Bryan S Sibert ◽  
Joseph Y Kim ◽  
Daniel Parrell ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Low Dose ◽  
Structural Information ◽  
Electron Tomography ◽  
Ion Beam ◽  
Three Dimensional ◽  
Structural Studies ◽  
Cryo Electron Tomography ◽  
Efficient Data

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here, we present robust tools for montage electron tomography tailored for vitrified specimens. The integration of correlative cryo-fluorescence microscopy, focused-ion beam milling, and micropatterning produces contextual three-dimensional architecture of cells. Montage tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

scholarly journals Cryo-ET reveals two major tubulin-based cytoskeleton structures in Toxoplasma gondii

2021 ◽  
Author(s):  
Stella Y. Sun ◽  
Li-av Segev-Zarko ◽  
Muyuan Chen ◽  
Grigore D. Pintilie ◽  
Michael F. Schmid ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Electron Tomography ◽  
Building Blocks ◽  
Molecular Structures ◽  
Accessory Proteins ◽  
Obligate Intracellular ◽  
Obligate Intracellular Parasite ◽  
Density Maps ◽  
Cryo Electron Tomography

In the obligate intracellular parasite, Toxoplasma gondii, the subpellicular microtubules (SPMTs) help maintain shape, while the apical conoid (also tubulin-based) is implicated in invasion. Here, we use cryo-electron tomography to determine the molecular structures of the SPMTs and the conoid-fibrils (CFs) in vitrified and detergent-lysed parasites. Subvolume densities from detergent-extracted parasites yielded averaged density maps at subnanometer resolutions, and these were related back to their architecture in situ. An intraluminal spiral (IS) lines the interior of the 13-protofilament SPMTs, revealing a preferred orientation of these microtubules relative to the parasite's long axis. Each CF is composed of 9 tubulin protofilaments, that produce a comma-shaped cross-section, plus additional associated components. Conoid protrusion, a crucial step in invasion, is associated with an altered pitch of each CF. The use of basic building blocks of protofilaments and different accessory proteins in one organism, illustrates the versatility of these critical structures.

scholarly journals High resolution in situ structural determination of heterogeneous specimen

2017 ◽  
Author(s):  
Benjamin A. Himes ◽  
Peijun Zhang
Keyword(s):  
Electron Tomography ◽  
Structural Determination ◽  
Viral Capsids ◽  
Link Type ◽  
Current State ◽  
Novel Approach ◽  
Cryo Electron Tomography ◽  
Insight Into

AbstractMacromolecular complexes are intrinsically flexible and often challenging to purify for structure determination by single particle cryoEM. Such complexes may be studied in situ using cryo-electron tomography combined with sub-tomogram alignment and classification, which in exceptional cases reaches sub-nanometer resolution, yielding insight into structure-function relationships. All maps currently deposited in the EMDB with resolution < 9 Å are from macromolecules that form ordered structural arrays, like viral capsids, which greatly simplifies structural determination. Extending this approach to more common specimens that exhibit conformational or compositional heterogeneity, and may be available in limited numbers, remains challenging. We developed emClarity, a GPU-accelerated image processing package, specifically to address fundamental hurdles to this aim, and demonstrate significant improvements in the resolution of maps compared to those generated using current state-of-the-art software. Furthermore, we devise a novel approach to sub-tomogram classification that reveals functional states not previously observed with the same data.The software is freely available from https://www.github.com/bHimes/emClarityTutorial documentation and videos at https://www.github.com/bHimes/emClarity/wiki

scholarly journals Recent Developments in the Determination of Biomarkers of Tobacco Smoke Exposure in Biological Specimens: A Review

2021 ◽  
Vol 18 (4) ◽  
pp. 1768
Author(s):  
Hernâni Marques ◽  
Pedro Cruz-Vicente ◽  
Tiago Rosado ◽  
Mário Barroso ◽  
Luís A. Passarinha ◽  
...  
Keyword(s):  
Tobacco Smoke ◽  
Solid Phase ◽  
Smoke Exposure ◽  
Tobacco Smoke Exposure ◽  
World Health ◽  
Second Hand Smoke ◽  
Tobacco Exposure ◽  
Wide Range ◽  
Recent Developments

Environmental tobacco smoke exposure (ETS) and smoking have been described as the most prevalent factors in the development of certain diseases worldwide. According to the World Health Organization, more than 8 million people die every year due to exposure to tobacco, around 7 million due to direct ETS and the remaining due to exposure to second-hand smoke. Both active and second-hand exposure can be measured and controlled using specific biomarkers of tobacco and its derivatives, allowing the development of more efficient public health policies. Exposure to these compounds can be measured using different methods (involving for instance liquid- or gas-chromatographic procedures) in a wide range of biological specimens to estimate the type and degree of tobacco exposure. In recent years, a lot of research has been carried out using different extraction methods and different analytical equipment; this way, liquid–liquid extraction, solid-phase extraction or even miniaturized procedures have been used, followed by chromatographic analysis coupled mainly to mass spectrometric detection. Through this type of methodologies, second-hand smokers can be distinguished from active smokers, and this is also valid for e-cigarettes and vapers, among others, using their specific biomarkers. This review will focus on recent developments in the determination of tobacco smoke biomarkers, including nicotine and other tobacco alkaloids, specific nitrosamines, polycyclic aromatic hydrocarbons, etc. The methods for their detection will be discussed in detail, as well as the potential use of threshold values to distinguish between types of exposure.

Cryo electron tomography of vitrified fibroblasts: Microtubule plus ends in situ

2008 ◽  
Vol 161 (3) ◽  
pp. 459-468 ◽  
Author(s):  
Roman I. Koning ◽  
Sandra Zovko ◽  
Montserrat Bárcena ◽  
Gert T. Oostergetel ◽  
Henk K. Koerten ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Cryo Electron Tomography
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