1,076 results match your criteria cryo-electron tomography


Structural evidence for extracellular silica formation by diatoms.

Nat Commun 2021 07 30;12(1):4639. Epub 2021 Jul 30.

Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel.

The silica cell wall of diatoms, a widespread group of unicellular microalgae, is an exquisite example for the ability of organisms to finely sculpt minerals under strict biological control. The prevailing paradigm for diatom silicification is that this is invariably an intracellular process, occurring inside specialized silica deposition vesicles that are responsible for silica precipitation and morphogenesis. Here, we study the formation of long silicified extensions that characterize many diatom species. Read More

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Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress.

Nat Commun 2021 07 30;12(1):4629. Epub 2021 Jul 30.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. Read More

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

Elife 2021 Jul 29;10. Epub 2021 Jul 29.

University of Zürich, Zurich, Switzerland.

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. Read More

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Mechanistic insight into bacterial entrapment by septin cage reconstitution.

Nat Commun 2021 07 23;12(1):4511. Epub 2021 Jul 23.

Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, UK.

Septins are cytoskeletal proteins that assemble into hetero-oligomeric complexes and sense micron-scale membrane curvature. During infection with Shigella flexneri, an invasive enteropathogen, septins restrict actin tail formation by entrapping bacteria in cage-like structures. Here, we reconstitute septin cages in vitro using purified recombinant septin complexes (SEPT2-SEPT6-SEPT7), and study how these recognize bacterial cells and assemble on their surface. Read More

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Never retire: An introduction to the Baumeister- Humphreys-Spence--Urban birthday issue.

Authors:
P W Hawkes

Ultramicroscopy 2021 May 24:113323. Epub 2021 May 24.

CEMES-CNRS, B.P. 94347, 31055 Toulouse Cedex (France). Electronic address:

The professional and private lives of Wolfgang Baumeister, Sir Colin Humphreys, John C.H. Spence and Knut Urban are retraced on the occasion of their 80th (Humphreys, Urban) and 75th (Baumeister, Spence) birthdays. Read More

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Structural basis of torque generation in the bi-directional bacterial flagellar motor.

Trends Biochem Sci 2021 Jul 19. Epub 2021 Jul 19.

Structural Biology of Molecular Machines Group, Protein Structure & Function Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address:

The flagellar stator unit is an oligomeric complex of two membrane proteins (MotAB) that powers bi-directional rotation of the bacterial flagellum. Harnessing the ion motive force across the cytoplasmic membrane, the stator unit operates as a miniature rotary motor itself to provide torque for rotation of the flagellum. Recent cryo-electron microscopic (cryo-EM) structures of the stator unit provided novel insights into its assembly, function, and subunit stoichiometry, revealing the ion flux pathway and the torque generation mechanism. Read More

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CryoSIM: super-resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultrastructural imaging.

Optica 2020 Jul 13;7(7):802-812. Epub 2020 Jul 13.

Micron Advanced Bio-imaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. Read More

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Structural and functional characterization of the intracellular filament-forming nitrite oxidoreductase multiprotein complex.

Nat Microbiol 2021 Jul 15. Epub 2021 Jul 15.

Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Heidelberg, Germany.

Nitrate is an abundant nutrient and electron acceptor throughout Earth's biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. Read More

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Structural analysis of the architecture and in situ localization of the main S-layer complex in Deinococcus radiodurans.

Structure 2021 Jul 12. Epub 2021 Jul 12.

Department of Plant Physiology, Warsaw University of Life Sciences - SGGW, 02-776 Warsaw, Poland; Laboratory of Plant Physiology and Photobiology, Department of Life and Environmental Sciences, University of Cagliari, 09123 Cagliari, Italy. Electronic address:

Bacterial surface layers are paracrystalline assemblies of proteins that provide the first line of defense against environmental shocks. Here, we report the 3D structure, in situ localization, and orientation of the S-layer deinoxanthin-binding complex (SDBC), a hetero-oligomeric assembly of proteins that in Deinococcus radiodurans represents the main S-layer unit. The SDBC is resolved at 11-Å resolution by single-particle analysis, while its in situ localization is determined by cryo-electron crystallography on intact cell-wall fragments leading to a projection map at 4. Read More

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Preparation of Doublet Microtubule Fraction for Single ParticleCryo-electron Microscopy.

Bio Protoc 2021 Jun 5;11(11):e4041. Epub 2021 Jun 5.

Department of Anatomy and Cell Biology, McGill University, Montréal, Canada.

Over the years, studying the ultrastructure of the eukaryotic cilia/flagella using electron microscopy (EM) has contributed significantly toward our understanding of ciliary function. Major complexes in the cilia, such as inner and outer dynein arms, radial spokes, and dynein regulatory complexes, were originally discovered by EM. Classical resin-embedding EM or cryo-electron tomography can be performed directly on the isolated cilia or in some cases, cilia directly attached to the cell body. Read More

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Cryo-electron tomography provides topological insights into mutant huntingtin exon 1 and polyQ aggregates.

Commun Biol 2021 07 8;4(1):849. Epub 2021 Jul 8.

Department of Bioengineering and James H. Clark Center, Stanford University, Stanford, CA, USA.

Huntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Read More

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Rotavirus research: 2014-2020.

Virus Res 2021 Jul 2:198499. Epub 2021 Jul 2.

Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK. Electronic address:

Rotaviruses are major causes of acute gastroenteritis in infants and young children worldwide and also cause disease in the young of many other mammalian and of avian species. During the recent 5-6 years rotavirus research has benefitted in a major way from the establishment of plasmid only-based reverse genetics systems, the creation of human and other mammalian intestinal enteroids, and from the wide application of structural biology (cryo-electron microscopy, cryo-EM tomography) and complementary biophysical approaches. All of these have permitted to gain new insights into structure-function relationships of rotaviruses and their interactions with the host. Read More

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Molecular-scale visualization of sarcomere contraction within native cardiomyocytes.

Nat Commun 2021 07 2;12(1):4086. Epub 2021 Jul 2.

Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.

Sarcomeres, the basic contractile units of striated muscle, produce the forces driving muscular contraction through cross-bridge interactions between actin-containing thin filaments and myosin II-based thick filaments. Until now, direct visualization of the molecular architecture underlying sarcomere contractility has remained elusive. Here, we use in situ cryo-electron tomography to unveil sarcomere contraction in frozen-hydrated neonatal rat cardiomyocytes. Read More

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Coming of Age: Cryo-Electron Tomography as a Versatile Tool to Generate High-Resolution Structures at Cellular/Biological Interfaces.

Int J Mol Sci 2021 Jun 8;22(12). Epub 2021 Jun 8.

Department of Biomedical Engineering and Health Systems, Royal Technical Institute (KTH), Hälsovägen 11C, 141 27 Huddinge, Sweden.

Over the last few years, cryo electron microscopy has become the most important method in structural biology. While 80% of deposited maps are from single particle analysis, electron tomography has grown to become the second most important method. In particular sub-tomogram averaging has matured as a method, delivering structures between 2 and 5 Å from complexes in cells as well as in vitro complexes. Read More

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VHUT-cryo-FIB, a method to fabricate frozen hydrated lamellae from tissue specimens for in situ cryo-electron tomography.

J Struct Biol 2021 Jun 24;213(3):107763. Epub 2021 Jun 24.

National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing, China; Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Physical Science Laboratory, Huairou National Comprehensive Science Center, No. 5 Yanqi East Second Street, Beijing 101400, China. Electronic address:

Cryo-electron tomography (cryo-ET) provides a promising approach to study intact structures of macromolecules in situ, but the efficient preparation of high-quality cryosections represents a bottleneck. Although cryo-focused ion beam (cryo-FIB) milling has emerged for large and flat cryo-lamella preparation, its application to tissue specimens remains challenging. Here, we report an integrated workflow, VHUT-cryo-FIB, for efficiently preparing frozen hydrated tissue lamella that can be readily used in subsequent cryo-ET studies. Read More

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Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity.

Cell 2021 Jul 23;184(14):3643-3659.e23. Epub 2021 Jun 23.

Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; Helmholtz Pioneer Campus, Helmholtz Zentrum München, 85764 Neuherberg, Germany; Department of Chemistry, Technical University of Munich, 85748 Garching, Germany. Electronic address:

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Read More

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Where in the cell is my protein?

Authors:
David J DeRosier

Q Rev Biophys 2021 Jun 21;54:e9. Epub 2021 Jun 21.

Brandeis University, Waltham, MA, USA.

The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization. Read More

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Composition and Biophysical Properties of the Sorting Platform Pods in the Type III Secretion System.

Front Cell Infect Microbiol 2021 3;11:682635. Epub 2021 Jun 3.

Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, United States.

, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisome's cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. Read More

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Cooperative multivalent receptor binding promotes exposure of the SARS-CoV-2 fusion machinery core.

bioRxiv 2021 Jun 7. Epub 2021 Jun 7.

The molecular events that permit the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bind, fuse, and enter cells are important to understand for both fundamental and therapeutic reasons. Spike proteins consist of S1 and S2 domains, which recognize angiotensin-converting enzyme 2 (ACE2) receptors and contain the viral fusion machinery, respectively. Ostensibly, the binding of spike trimers to ACE2 receptors promotes the preparation of the fusion machinery by dissociation of the S1 domains. Read More

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Soft-matter properties of multilayer chromosomes.

Authors:
Joan-Ramon Daban

Phys Biol 2021 Jul 12;18(5). Epub 2021 Jul 12.

Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193-Bellaterra (Barcelona), Spain.

This perspective aims to identify the relationships between the structural and dynamic properties of chromosomes and the fundamental properties of soft-matter systems. Chromatin is condensed into metaphase chromosomes during mitosis. The resulting structures are elongated cylinders having micrometer-scale dimensions. Read More

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3D-visualization of amyloid-β oligomer interactions with lipid membranes by cryo-electron tomography.

Chem Sci 2021 Mar 31;12(20):6896-6907. Epub 2021 Mar 31.

School of Biological and Chemical Sciences, Queen Mary University of London Mile End Road London E1 4NS UK

Amyloid-β (Aβ) assemblies have been shown to bind to lipid bilayers. This can disrupt membrane integrity and cause a loss of cellular homeostasis, that triggers a cascade of events leading to Alzheimer's disease. However, molecular mechanisms of Aβ cytotoxicity and how the different assembly forms interact with the membrane remain enigmatic. Read More

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Maintenance of complex I and its supercomplexes by NDUF-11 is essential for mitochondrial structure, function and health.

J Cell Sci 2021 Jul 9;134(13). Epub 2021 Jul 9.

School of Biochemistry , University of Bristol, Bristol BS8 1TD, UK.

Mitochondrial supercomplexes form around a conserved core of monomeric complex I and dimeric complex III; wherein a subunit of the former, NDUFA11, is conspicuously situated at the interface. We identified nduf-11 (B0491.5) as encoding the Caenorhabditis elegans homologue of NDUFA11. Read More

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HEMNMA-3D: Cryo Electron Tomography Method Based on Normal Mode Analysis to Study Continuous Conformational Variability of Macromolecular Complexes.

Front Mol Biosci 2021 19;8:663121. Epub 2021 May 19.

IMPMC-UMR 7590 CNRS, Sorbonne Université, Muséum National d'Histoire Naturelle, Paris, France.

Cryogenic electron tomography (cryo-ET) allows structural determination of biomolecules in their native environment (). Its potential of providing information on the dynamics of macromolecular complexes in cells is still largely unexploited, due to the challenges of the data analysis. The crowded cell environment and continuous conformational changes of complexes make difficult disentangling the data heterogeneity. Read More

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Interphase epichromatin: last refuge for the 30-nm chromatin fiber?

Chromosoma 2021 Jun 5. Epub 2021 Jun 5.

Department of Pharmaceutical Sciences and Administration, School of Pharmacy, University of New England, 716 Stevens Avenue, Portland, ME, 04103, USA.

"Interphase epichromatin" describes the surface of chromatin located adjacent to the interphase nuclear envelope. It was discovered in 2011 using a bivalent anti-nucleosome antibody (mAb PL2-6), now known to be directed against the nucleosome acidic patch. The molecular structure of interphase epichromatin is unknown, but is thought to be heterochromatic with a high density of "exposed" acidic patches. Read More

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Studying Reaction Mechanisms in Solution Using a Distributed Electron Microscopy Method.

ACS Nano 2021 06 2;15(6):10296-10308. Epub 2021 Jun 2.

Department of Chemistry, University of California, Irvine, Irvine, California 92697-2025, United States.

Electron microscopy (EM) of materials undergoing chemical reactions provides knowledge of the underlying mechanisms. However, the mechanisms are often complex and cannot be fully resolved using a single method. Here, we present a distributed electron microscopy method for studying complex reactions. Read More

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Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer.

Nat Commun 2021 05 28;12(1):3226. Epub 2021 May 28.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.

Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Read More

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A phase-separated nuclear GBPL circuit controls immunity in plants.

Nature 2021 Jun 26;594(7863):424-429. Epub 2021 May 26.

Howard Hughes Medical Institute, New Haven, CT, USA.

Liquid-liquid phase separation (LLPS) has emerged as a central paradigm for understanding how membraneless organelles compartmentalize diverse cellular activities in eukaryotes. Here we identify a superfamily of plant guanylate-binding protein (GBP)-like GTPases (GBPLs) that assemble LLPS-driven condensates within the nucleus to protect against infection and autoimmunity. In Arabidopsis thaliana, two members of this family-GBPL1 and GBPL3-undergo phase-transition behaviour to control transcriptional responses as part of an allosteric switch that is triggered by exposure to biotic stress. Read More

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Asymmetric localization of the cell division machinery during sporulation.

Elife 2021 May 21;10. Epub 2021 May 21.

Division of Biological Sciences, University of California San Diego, La Jolla, United States.

The Gram-positive bacterium can divide via two modes. During vegetative growth, the division septum is formed at the midcell to produce two equal daughter cells. However, during sporulation, the division septum is formed closer to one pole to yield a smaller forespore and a larger mother cell. Read More

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Integrating on-grid immunogold labeling and cryo-electron tomography to reveal photosystem II structure and spatial distribution in thylakoid membranes.

J Struct Biol 2021 May 16;213(3):107746. Epub 2021 May 16.

Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, United States; Institute for Quantitative Biomedicine, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, United States. Electronic address:

A long-standing challenge in cell biology is elucidating the structure and spatial distribution of individual membrane-bound proteins, protein complexes and their interactions in their native environment. Here, we describe a workflow that combines on-grid immunogold labeling, followed by cryo-electron tomography (cryoET) imaging and structural analyses to identify and characterize the structure of photosystem II (PSII) complexes. Using an antibody specific to a core subunit of PSII, the D1 protein (uniquely found in the water splitting complex in all oxygenic photoautotrophs), we identified PSII complexes in biophysically active thylakoid membranes isolated from a model marine diatom Phaeodactylum tricornutum. Read More

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Current data processing strategies for cryo-electron tomography and subtomogram averaging.

Biochem J 2021 May;478(10):1827-1845

Institute of Structural and Molecular Biology, Birkbeck College, Malet St., London WC1E 7HX, U.K.

Cryo-electron tomography (cryo-ET) can be used to reconstruct three-dimensional (3D) volumes, or tomograms, from a series of tilted two-dimensional images of biological objects in their near-native states in situ or in vitro. 3D subvolumes, or subtomograms, containing particles of interest can be extracted from tomograms, aligned, and averaged in a process called subtomogram averaging (STA). STA overcomes the low signal to noise ratio within the individual subtomograms to generate structures of the particle(s) of interest. Read More

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