scholarly journals Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Miriam S Weber ◽  
Matthias Eibauer ◽  
Suganya Sivagurunathan ◽  
Thomas M Magin ◽  
Robert D Goldman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Structural Heterogeneity ◽  
Epithelial Tissue ◽  
Cell Periphery ◽  
Cryo Electron Microscopy ◽  
Dimensional Classification ◽  
Keratin Filaments ◽  
Electron Dense Core ◽  
Subunit Organization

Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell periphery, which provides resistance to mechanical stresses. Mutations in keratin genes are related to a variety of epithelial tissue diseases. Despite their importance, the molecular structure of keratin filaments remains largely unknown. In this study, we analyzed the structure of keratin 5/keratin 14 filaments within ghost mouse keratinocytes by cryo-electron microscopy and cryo-electron tomography. By averaging a large number of keratin segments, we have gained insights into the helical architecture of the filaments. Two-dimensional classification revealed profound variations in the diameter of keratin filaments and their subunit organization. Computational reconstitution of filaments of substantial length uncovered a high degree of internal heterogeneity along single filaments, which can contain regions of helical symmetry, regions with less symmetry and regions with significant diameter fluctuations. Cross section views of filaments revealed that keratins form hollow cylinders consisting of multiple protofilaments, with an electron dense core located in the center of the filament. These findings shed light on the complex and remarkable heterogenic architecture of keratin filaments, suggesting that they are highly flexible, dynamic cytoskeletal structures.

scholarly journals Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo-Electron Microscopy Applications

2013 ◽  
Vol 20 (1) ◽  
pp. 164-174 ◽  
Author(s):  
Gabriella Kiss ◽  
Xuemin Chen ◽  
Melinda A. Brindley ◽  
Patricia Campbell ◽  
Claudio L. Afonso ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Measles Virus ◽  
Influenza A ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Nitrilotriacetic Acid ◽  
Dimensional Structure ◽  
Enveloped Viruses ◽  
Cryo Electron Microscopy ◽  
Human Immunodeficiency Virus Virus

AbstractElectron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.

Frontiers in Cryo Electron Microscopy of Complex Macromolecular Assemblies

2019 ◽  
Vol 21 (1) ◽  
pp. 395-415 ◽  
Author(s):  
Jana Ognjenović ◽  
Reinhard Grisshammer ◽  
Sriram Subramaniam
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Specimen Preparation ◽  
Macromolecular Assemblies ◽  
Cryo Electron Microscopy ◽  
G Protein Coupled ◽  
Improved Methods

In recent years, cryo electron microscopy (cryo-EM) technology has been transformed with the development of better instrumentation, direct electron detectors, improved methods for specimen preparation, and improved software for data analysis. Analyses using single-particle cryo-EM methods have enabled determination of structures of proteins with sizes smaller than 100 kDa and resolutions of ∼2 Å in some cases. The use of electron tomography combined with subvolume averaging is beginning to allow the visualization of macromolecular complexes in their native environment in unprecedented detail. As a result of these advances, solutions to many intractable challenges in structural and cell biology, such as analysis of highly dynamic soluble and membrane-embedded protein complexes or partially ordered protein aggregates, are now within reach. Recent reports of structural studies of G protein–coupled receptors, spliceosomes, and fibrillar specimens illustrate the progress that has been made using cryo-EM methods, and are the main focus of this review.

scholarly journals Structural heterogeneity of apoB-containing serum lipoproteins visualized using cryo-electron microscopy

1999 ◽  
Vol 40 (10) ◽  
pp. 1827-1836
Author(s):  
Rik van Antwerpen ◽  
Michael La Belle ◽  
Edita Navratilova ◽  
Ronald M. Krauss
Keyword(s):  
Electron Microscopy ◽  
Structural Heterogeneity ◽  
Serum Lipoproteins ◽  
Cryo Electron Microscopy

scholarly journals Modern Uses of Electron Microscopy for Detection of Viruses

2009 ◽  
Vol 22 (4) ◽  
pp. 552-563 ◽  
Author(s):  
Cynthia S. Goldsmith ◽  
Sara E. Miller
Keyword(s):  
Electron Microscopy ◽  
Immunoelectron Microscopy ◽  
Electron Tomography ◽  
Emerging Diseases ◽  
Structural Components ◽  
Cryo Electron Microscopy ◽  
Cellular Machinery ◽  
Diagnostic Setting

SUMMARY Electron microscopy, considered by some to be an old technique, is still on the forefront of both clinical viral diagnoses and viral ultrastructure and pathogenesis studies. In the diagnostic setting, it is particularly valuable in the surveillance of emerging diseases and potential bioterrorism viruses. In the research arena, modalities such as immunoelectron microscopy, cryo-electron microscopy, and electron tomography have demonstrated how viral structural components fit together, attach to cells, assimilate during replication, and associate with the cellular machinery during replication and egression. These studies provide information for treatment and vaccine strategies.

scholarly journals Nanoscale characterization of the biomolecular corona by cryo-electron microscopy, cryo-electron tomography, and image simulation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sara Sheibani ◽  
Kaustuv Basu ◽  
Ali Farnudi ◽  
Aliakbar Ashkarran ◽  
Muneyoshi Ichikawa ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Three Dimensional ◽  
True Nature ◽  
Cryo Electron Microscopy ◽  
Wide Range ◽  
Cryo Electron Tomography ◽  
Nanoscale Characterization ◽  
Dimensional Reconstruction

AbstractThe biological identity of nanoparticles (NPs) is established by their interactions with a wide range of biomolecules around their surfaces after exposure to biological media. Understanding the true nature of the biomolecular corona (BC) in its native state is, therefore, essential for its safe and efficient application in clinical settings. The fundamental challenge is to visualize the biomolecules within the corona and their relationship/association to the surface of the NPs. Using a synergistic application of cryo-electron microscopy, cryo-electron tomography, and three-dimensional reconstruction, we revealed the unique morphological details of the biomolecules and their distribution/association with the surface of polystyrene NPs at a nanoscale resolution. The analysis of the BC at a single NP level and its variability among NPs in the same sample, and the discovery of the presence of nonspecific biomolecules in plasma residues, enable more precise characterization of NPs, improving predictions of their safety and efficacies.

scholarly journals Three-Dimensional Structure of Cytochrome c Nitrite Reductase As Determined by Cryo-Electron Microscopy

Acta Naturae ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 48-56 ◽  
Author(s):  
T. N. Baymukhametov ◽  
Yu. M. Chesnokov ◽  
E. B. Pichkur ◽  
K. M. Boyko ◽  
T. V. Tikhonova ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cytochrome C ◽  
Nitrite Reductase ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Side Chain ◽  
X Ray Diffraction ◽  
Cytochrome C Nitrite Reductase ◽  
Cryo Electron Microscopy

The structure of cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens was determined by cryo-electron microscopy (cryo-EM) at a 2.56 resolution. Possible structural heterogeneity of the enzyme was assessed. The backbone and side-chain orientations in the cryo-EM-based model are, in general, similar to those in the high-resolution X-ray diffraction structure of this enzyme.

scholarly journals 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy

2020 ◽  
Author(s):  
Florian Fäßler ◽  
Bettina Zens ◽  
Robert Hauschild ◽  
Florian K.M. Schur
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Electron Tomography ◽  
Low Cost ◽  
Ion Beam ◽  
Control Cell ◽  
Specimen Preparation ◽  
Micro Patterning ◽  
Cryo Electron Microscopy ◽  
3D Printed

AbstractCryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications.

Cryo Electron Microscopy of SSV1 phage particles

1995 ◽  
Vol 53 ◽  
pp. 842-843
Author(s):  
U. Ziese ◽  
D. Typke ◽  
R. Hegerl ◽  
W. Baumeister
Keyword(s):  
Electron Microscopy ◽  
Periodic Structure ◽  
Low Dose ◽  
Electron Tomography ◽  
Structural Data ◽  
Mass Determination ◽  
The Third ◽  
Negative Stain ◽  
Cryo Electron Microscopy ◽  
Thermophilic Archaeon

SSVl phages are lemon-shaped particles, normally about 90 nm x 40 nm in size, with short tail fibres attached to one pole, produced by the thermophilic archaeon Sulfolobus shibate, isolate B12. They are made of 3 different proteins and DNA (15.5 kbp). Two proteins, together with host lipid, form the envelope, the third protein is associated with the DNA. Interestingly, this virus produces particles of varying size and shape. We have investigated the mass of the virions by STEM mass determination, the inner structure by cryo-electron microscopy, and the shape variability by electron tomography. Automatic electron tomography (AET) has been shown to be a useful technique for collecting 3D structural data of individual biological particles under low dose conditions, in negative stain as well as in frozen-hydrated preparations.Taking electron micrographs of vitrified samples we obtained images revealing some details of the inner structure and, on some particles, a periodic structure of the envelope. (Fig. 1a-b) The inner structure has periodicities of about 2.5 nm, which is in agreement with that found by Lepault et al on vitrified samples of the phages lambda and T4.

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