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cryo electron tomography

Huan-Yuan Chen, Dapi Meng-Lin Chiang, Zi-Jing Lin, Chia-Chun Hsieh, Gung-Chian Yin, I-Chun Weng, Peter Guttermann, Stephan Werner, Katja Henzler, Gerd Schneider, Lee-Jene Lai, Fu-Tong Liu
Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation...
October 17, 2016: Scientific Reports
Vladan Lučić, Rubén Fernández-Busnadiego, Ulrike Laugks, Wolfgang Baumeister
Molecular complexes, arguably the basic units carrying cellular function, can be visualized directly in their native environment by cryo-electron tomography. Here we describe a procedure for the detection of small, pleomorphic membrane-bound molecular complexes in cryo-tomograms by a hierarchical connectivity segmentation. Validation on phantom and real data showed above 90% true positive rates. This segmentation procedure is implemented in the Pyto software package, together with methods for quantitative characterization and classification of complexes detected by our segmentation procedure and for statistical analysis between experimental conditions...
October 11, 2016: Journal of Structural Biology
Kazuyoshi Murata, Sayuri Hagiwara, Yoshitaka Kimori, Yasuko Kaneko
Some cyanobacteria exhibit compaction of DNA in synchrony with their circadian rhythms accompanying cell division. Since the structure is transient, it has not yet been described in detail. Here, we successfully visualize the ultrastructure of compacted DNA in the cyanobacterium Synechococcus elongatus PCC 7942 under rigorous synchronized cultivation by means of high-voltage cryo-electron tomography. In 3D reconstructions of rapidly frozen cells, the compacted DNA appears as an undulating rod resembling a eukaryotic condensed chromosome...
October 12, 2016: Scientific Reports
Andrea D Merg, Jennifer C Boatz, Abhishek Mandal, Gongpu Zhao, Soumitra Mokashi-Punekar, Chong Liu, Xianting Wang, Peijun Zhang, Patrick C A van der Wel, Nathaniel L Rosi
Chiral nanoparticle assemblies are an interesting class of materials whose chiroptical properties make them attractive for a variety of applications. Here, C18-(PEPAu(M-ox))2 (PEPAu(M-ox) = AYSSGAPPM(ox)PPF) is shown to direct the assembly of single-helical gold nanoparticle superstructures that exhibit exceptionally strong chiroptical activity at the plasmon frequency with absolute g-factor values up to 0.04. Transmission electron microscopy (TEM) and cryogenic electron tomography (cryo-ET) results indicate that the single helices have a periodic pitch of approximately 100 nm and consist of oblong gold nanoparticles...
October 11, 2016: Journal of the American Chemical Society
Long Pei, Min Xu, Zachary Frazier, Frank Alber
BACKGROUND: Cryo-electron tomography is an important tool to study structures of macromolecular complexes in close to native states. A whole cell cryo electron tomogram contains structural information of all its macromolecular complexes. However, extracting this information remains challenging, and relies on sophisticated image processing, in particular for template-free particle extraction, classification and averaging. To develop these methods it is crucial to realistically simulate tomograms of crowded cellular environments, which can then serve as ground truth models for assessing and optimizing methods for detection of complexes in cell tomograms...
October 5, 2016: BMC Bioinformatics
Matthias Ochs, Lars Knudsen, Jan Hegermann, Christoph Wrede, Roman Grothausmann, Christian Mühlfeld
In the nineteenth century, there was a dispute about the existence of a lung alveolar epithelium which remained unsolved until the invention of electron microscopy (EM) and its application to the lung. From the early 1960s, Ewald Weibel became the master of lung EM. He showed that the alveolar epithelium is covered with a lining layer containing surfactant. Weibel also explained the phenomenon of "non-nucleated plates" observed already in 1881 by Albert Kölliker. Weibel's most significant contribution was to the development of stereological methods...
September 29, 2016: Histochemistry and Cell Biology
Tanmay A M Bharat, Sjors H W Scheres
Electron cryo-tomography (cryo-ET) is a technique that is used to produce 3D pictures (tomograms) of complex objects such as asymmetric viruses, cellular organelles or whole cells from a series of tilted electron cryo-microscopy (cryo-EM) images. Averaging of macromolecular complexes found within tomograms is known as subtomogram averaging, and this technique allows structure determination of macromolecular complexes in situ. Subtomogram averaging is also gaining in popularity for the calculation of initial models for single-particle analysis...
November 2016: Nature Protocols
Peter Rez, Thomas Larsen, Michael Elbaum
Scanning transmission electron microscope (STEM) imaging has recently been applied to the cryo-tomography of thick biological specimens. As previously shown for plastic sections, STEM has a number of advantages for cryo-imaging compared to conventional wide-field TEM imaging. STEM is insensitive to phase coherence and is therefore suitable for much thicker specimens than TEM. Imaging in focus, with a long depth of field, also circumvents the complications of an oscillatory contrast transfer function and missing information at low spatial frequencies...
September 24, 2016: Journal of Structural Biology
Martin Beck, Wolfgang Baumeister
Traditionally, macromolecular structure determination is performed ex situ, that is, with purified materials. But, there are strong incentives to develop approaches to study them in situ in their native functional context. In recent years, cryo-electron tomography (cryo-ET) has emerged as a powerful method for visualizing the molecular organization of unperturbed cellular landscapes with the potential to attain near-atomic resolution. Here, we review recent work on several macromolecular assemblies, demonstrating the power of in situ studies...
September 23, 2016: Trends in Cell Biology
Thomas H Sharp, Frank G A Faas, Abraham J Koster, Piet Gros
Phase plates in cryo-electron tomography (cryoET) improve contrast, increasing the ability to discern separate molecules and molecular complexes in dense biomolecular environments. Here, we applied this new technology to the activation of the human complement system. Binding of C1 to antigen-antibody complexes initiates a cascade of proteolytic events that deposits molecules onto adjacent surfaces and terminates with the formation of membrane-attack-complex (MAC) pores in the targeted membranes. We imaged steps in this process using a Volta phase plate mounted on a Titan Krios equipped with a Falcon-II direct electron detector...
September 20, 2016: Journal of Structural Biology
Ruchao Peng, Tengfei Zhu, Babayemi Olawale Oladejo, Abednego Moki Musyoki, Yingzi Cui, Yi Shi, Peiyi Wang, George Fu Gao
Ebola virus (EBOV) harbors an RNA genome encapsidated by nucleoprotein (NP) along with other viral proteins to form a nucleocapsid complex. Previous Cryo-eletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP (NPΔ451-739) alone is capable of forming a helical nucleocapsid-like complex (NLC). However, the structural basis for NP-NP interaction and the dynamic procedure of the nucleocapsid assembly is yet poorly understood...
September 20, 2016: Protein & Cell
Vahid R Adineh, Ross K W Marceau, Tony Velkov, Jian Li, Jing Fu
Emergence of multidrug resistant Gram-negative bacteria has caused a global health crisis and last-line class of antibiotics such as polymyxins are increasingly used. The chemical composition at the cell surface plays a key role in antibiotic resistance. Unlike imaging the cellular ultrastructure with well-developed electron microscopy, the acquisition of a high-resolution chemical map of the bacterial surface still remains a technological challenge. In this study, we developed an atom probe tomography (APT) analysis approach to acquire mass spectra in the pulsed-voltage mode and reconstructed the 3D chemical distribution of atoms and molecules in the subcellular domain at the near-atomic scale...
October 14, 2016: Nano Letters
Audrey Guesdon, Franck Bazile, Rubén M Buey, Renu Mohan, Solange Monier, Ruddi Rodríguez García, Morgane Angevin, Claire Heichette, Ralph Wieneke, Robert Tampé, Laurence Duchesne, Anna Akhmanova, Michel O Steinmetz, Denis Chrétien
EB1 is a microtubule plus-end tracking protein that recognizes GTP-tubulin dimers in microtubules and thus represents a unique probe to investigate the architecture of the GTP cap of growing microtubule ends. Here, we conjugated EB1 to gold nanoparticles (EB1-gold) and imaged by cryo-electron tomography its interaction with dynamic microtubules assembled in vitro from purified tubulin. EB1-gold forms comets at the ends of microtubules assembled in the presence of GTP, and interacts with the outer surface of curved and straight tubulin sheets as well as closed regions of the microtubule lattice...
October 2016: Nature Cell Biology
James H Hurley, Eva Nogales
Autophagy is the process whereby cytosol, organelles, and inclusions are taken up in a double-membrane vesicle known as the autophagosome, and transported to the lysosome for destruction and recycling. Electron microscopy (EM) led to the discovery of autophagy in the 1950s and has been a central part of its characterization ever since. New capabilities in single particle EM studies of the autophagy machinery are beginning to provide exciting insights into the mechanisms of autophagosome initiation, growth, and substrate targeting...
September 7, 2016: Current Opinion in Structural Biology
Petr Chlanda, Elena Mekhedov, Hang Waters, Cindi L Schwartz, Elizabeth R Fischer, Rolf J Ryham, Fredric S Cohen, Paul S Blank, Joshua Zimmerberg
Influenza A virus haemagglutinin conformational change drives the membrane fusion of viral and endosomal membranes at low pH. Membrane fusion proceeds through an intermediate called hemifusion(1,2). For viral fusion, the hemifusion structures are not determined(3). Here, influenza virus-like particles(4) carrying wild-type haemagglutinin or haemagglutinin hemifusion mutant G1S(5) and liposome mixtures were studied at low pH by Volta phase plate cryo-electron tomography, which improves the signal-to-noise ratio close to focus...
2016: Nature Microbiology
W Wan, J A G Briggs
Cryo-electron tomography (cryo-ET) allows 3D volumes to be reconstructed from a set of 2D projection images of a tilted biological sample. It allows densities to be resolved in 3D that would otherwise overlap in 2D projection images. Cryo-ET can be applied to resolve structural features in complex native environments, such as within the cell. Analogous to single-particle reconstruction in cryo-electron microscopy, structures present in multiple copies within tomograms can be extracted, aligned, and averaged, thus increasing the signal-to-noise ratio and resolution...
2016: Methods in Enzymology
Yadong Yu, Yu-Lin Kuang, Dongsheng Lei, Xiaobo Zhai, Meng Zhang, Ronald M Krauss, Gang Ren
Human VLDLs assembled in the liver and secreted into the circulation supply energy to peripheral tissues. VLDL lipolysis yields atherogenic LDLs and VLDL remnants that strongly correlate with CVD. Although the composition of VLDL particles has been well-characterized, their 3D structure is elusive because of their variations in size, heterogeneity in composition, structural flexibility, and mobility in solution. Here, we employed cryo-electron microscopy and individual-particle electron tomography to study the 3D structure of individual VLDL particles (without averaging) at both below and above their lipid phase transition temperatures...
October 2016: Journal of Lipid Research
Hongli Hu, Xing Meng
Rabbit muscle vesicles derived from sarcoplasmic reticulum were used as a material in studying networks of ryanodine receptors by cryo electron tomography. Three-dimensional analysis reveals the dynamical features of these networks. It was found that the connection angles were rotated along the transmembrane axis of ryanodine receptors. Majority of the connections was observed at domains 6/6 of ryanodine receptors while a small group of connections were showed at domains 9/10. The flexible rotation and connection shift seem to facilitate the extension of an annular network on the wall of the sarcoplasmic reticulum in a triad...
June 13, 2016: European Journal of Translational Myology
Miroslava Schaffer, Julia Mahamid, Benjamin D Engel, Tim Laugks, Wolfgang Baumeister, Jürgen M Plitzko
While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET...
July 19, 2016: Journal of Structural Biology
Florian K M Schur, Martin Obr, Wim J H Hagen, William Wan, Arjen J Jakobi, Joanna M Kirkpatrick, Carsten Sachse, Hans-Georg Kräusslich, John A G Briggs
Immature HIV-1 assembles at and buds from the plasma membrane before proteolytic cleavage of the viral Gag polyprotein induces structural maturation. Maturation can be blocked by maturation inhibitors (MIs), thereby abolishing infectivity. The CA (capsid) and SP1 (spacer peptide 1) region of Gag is the key regulator of assembly and maturation and is the target of MIs. We applied optimized cryo-electron tomography and subtomogram averaging to resolve this region within assembled immature HIV-1 particles at 3...
July 29, 2016: Science
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