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Cryo electron microscopy

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https://www.readbyqxmd.com/read/29166561/structural-characterization-of-native-and-modified-encapsulins-as-nanoplatforms-for-in-vitro-catalysis-and-cellular-uptake
#1
Rindia M Putri, Carolina Allende-Ballestero, Daniel Luque, Robin Klem, Katerina-Asteria Rousou, Aijie Liu, Christoph H-H Traulsen, W Frederik Rurup, Melissa S T Koay, Jose R Castón, Jeroen J L M Cornelissen
Recent years have witnessed the emergence of bacterial semi-organelle encapsulins as promising platforms for bio-nanotechnology. To advance the development of encapsulins as nanoplatforms, a functional and structural basis of these assemblies is required. Encapsulin from Brevibacterium linens is known to be a protein-based vessel for an enzyme cargo in its cavity, which could be replaced with a foreign cargo resulting in a modified encapsulin. Here, we characterize the native structure of B. linens encapsulins with both native and foreign cargo using cryo-electron microscopy (cryo-EM)...
November 22, 2017: ACS Nano
https://www.readbyqxmd.com/read/29163452/the-opportunistic-pathogen-vibrio-vulnificus-produces-outer-membrane-vesicles-in-a-spatially-distinct-manner-related-to-capsular-polysaccharide
#2
Cheri M Hampton, Ricardo C Guerrero-Ferreira, Rachel E Storms, Jeannette V Taylor, Hong Yi, Paul A Gulig, Elizabeth R Wright
Vibrio vulnificus, a bacterial species that inhabits brackish waters, is an opportunistic pathogen of humans. V. vulnificus infections can cause acute gastroenteritis, invasive septicemia, tissue necrosis, and potentially death. Virulence factors associated with V. vulnificus include the capsular polysaccharide (CPS), lipopolysaccharide, flagellum, pili, and outer membrane vesicles (OMVs). The aims of this study were to characterize the morphology of V. vulnificus cells and the formation and arrangement of OMVs using cryo-electron microscopy (cryo-EM)...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/29155784/correlative-super-resolution-and-electron-microscopy-to-resolve-protein-localization-in-zebrafish-retina
#3
José M Mateos, Gery Barmettler, Jana Doehner, Irene Ojeda Naharros, Bruno Guhl, Stephan C F Neuhauss, Andres Kaech, Ruxandra Bachmann-Gagescu, Urs Ziegler
We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM)...
November 10, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29150609/atomic-resolution-snapshot-of-leishmania-ribosome-inhibition-by-the-aminoglycoside-paromomycin
#4
Moran Shalev-Benami, Yan Zhang, Haim Rozenberg, Yuko Nobe, Masato Taoka, Donna Matzov, Ella Zimmerman, Anat Bashan, Toshiaki Isobe, Charles L Jaffe, Ada Yonath, Georgios Skiniotis
Leishmania is a single-celled eukaryotic parasite afflicting millions of humans worldwide, with current therapies limited to a poor selection of drugs that mostly target elements in the parasite's cell envelope. Here we determined the atomic resolution electron cryo-microscopy (cryo-EM) structure of the Leishmania ribosome in complex with paromomycin (PAR), a highly potent compound recently approved for treatment of the fatal visceral leishmaniasis (VL). The structure reveals the mechanism by which the drug induces its deleterious effects on the parasite...
November 17, 2017: Nature Communications
https://www.readbyqxmd.com/read/29149017/the-ms6-mycolyl-arabinogalactan-esterase-lysb-is-essential-for-an-efficient-mycobacteriophage-induced-lysis
#5
Adriano M Gigante, Cheri M Hampton, Rebecca S Dillard, Filipa Gil, Maria João Catalão, José Moniz-Pereira, Elizabeth R Wright, Madalena Pimentel
All dsDNA phages encode two proteins involved in host lysis, an endolysin and a holin that target the peptidoglycan and cytoplasmic membrane, respectively. Bacteriophages that infect Gram-negative bacteria encode additional proteins, the spanins, involved in disruption of the outer membrane. Recently, a gene located in the lytic cassette was identified in the genomes of mycobacteriophages, which encodes a protein (LysB) with mycolyl-arabinogalactan esterase activity. Taking in consideration the complex mycobacterial cell envelope that mycobacteriophages encounter during their life cycle, it is valuable to evaluate the role of these proteins in lysis...
November 17, 2017: Viruses
https://www.readbyqxmd.com/read/29146813/structural-basis-of-bacterial-transcription-activation
#6
Bin Liu, Chuan Hong, Rick K Huang, Zhiheng Yu, Thomas A Steitz
In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3',5'-monophosphate receptor protein (CAP), a classic activator, is able to activate transcription independently through two different mechanisms. Understanding the class I mechanism requires an intact transcription activation complex (TAC) structure at a high resolution. Here we report a high-resolution cryo-electron microscopy structure of an intact Escherichia coli class I TAC containing a CAP dimer, a σ(70)-RNA polymerase (RNAP) holoenzyme, a complete class I CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide...
November 17, 2017: Science
https://www.readbyqxmd.com/read/29146805/the-structural-basis-of-flagellin-detection-by-naip5-a-strategy-to-limit-pathogen-immune-evasion
#7
Jeannette L Tenthorey, Nicole Haloupek, José Ramón López-Blanco, Patricia Grob, Elise Adamson, Ella Hartenian, Nicholas A Lind, Natasha M Bourgeois, Pablo Chacón, Eva Nogales, Russell E Vance
Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form a multiprotein complex called an inflammasome. Here we report the cryo-electron microscopy structure of the assembled ~1...
November 17, 2017: Science
https://www.readbyqxmd.com/read/29144446/structure-and-assembly-of-the-ebola-virus-nucleocapsid
#8
William Wan, Larissa Kolesnikova, Mairi Clarke, Alexander Koehler, Takeshi Noda, Stephan Becker, John A G Briggs
Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever. Filoviruses are within the order Mononegavirales, which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical nucleocapsid. The nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication...
November 16, 2017: Nature
https://www.readbyqxmd.com/read/29143694/cryo-scanning-electron-microscopy-sem-and-scanning-transmission-electron-microscopy-stem-in-sem-for-bio-and-organo-mineral-interface-characterization-in-the-environment
#9
Guillaume Wille, Jennifer Hellal, Patrick Ollivier, Annie Richard, Agnes Burel, Louis Jolly, Marc Crampon, Caroline Michel
Understanding biofilm interactions with surrounding substratum and pollutants/particles can benefit from the application of existing microscopy tools. Using the example of biofilm interactions with zero-valent iron nanoparticles (nZVI), this study aims to apply various approaches in biofilm preparation and labeling for fluorescent or electron microscopy and energy dispersive X-ray spectrometry (EDS) microanalysis for accurate observations. According to the targeted microscopy method, biofilms were sampled as flocs or attached biofilm, submitted to labeling using 4',6-diamidino-2-phenylindol, lectins PNA and ConA coupled to fluorescent dye or gold nanoparticles, and prepared for observation (fixation, cross-section, freezing, ultramicrotomy)...
November 16, 2017: Microscopy and Microanalysis
https://www.readbyqxmd.com/read/29140718/pem-fitter-a-coarse-grained-method-to-validate-protein-candidate-models
#10
Kamal Al Nasr, Christopher Jones, Feras Yousef, Ruba Jebril
The volumetric images produced by Cryo-Electron Microscopy (cryo-EM) technique are used to model macromolecular assemblies and machines. De novo protein modeling uses these images to computationally model the structure of the molecules. Many candidate conformations are usually generated during the intermediate step. Conventionally, each of these candidates is evaluated by time-consuming approaches such as potential energy. We introduce an initial version of a geometrical screening method that uses the skeleton of the cryo-EM images to evaluate candidate structures...
November 15, 2017: Journal of Computational Biology: a Journal of Computational Molecular Cell Biology
https://www.readbyqxmd.com/read/29135688/membrane-microdomains-and-the-regulation-of-hdl-biogenesis
#11
Jacques Genest, Adel Schwertani, Hong Y Choi
PURPOSE OF REVIEW: The major cardio-protective function of HDL is to remove excess cellular cholesterol in the process of HDL particle formation and maturation. The HDL biogenic procedure requiring protein-lipid interactions has been incompletely understood, and here we discuss recent progress and insights into the mechanism of HDL biogenesis. RECENT FINDINGS: The initial and rate-limiting step of HDL biogenesis is the interaction between apoA-I and plasma membrane microdomains created by ATP-binding cassette transporter A1 (ABCA1) transporter...
November 11, 2017: Current Opinion in Lipidology
https://www.readbyqxmd.com/read/29132829/the-lipolytic-degradation-of-highly-structured-cubic-micellar-nanoparticles-of-soy-phosphatidylcholine-and-glycerol-dioleate-by-phospholipase-a2-and-triacylglycerol-lipase
#12
Maria Wadsäter, Justas Barauskas, Fredrik Tiberg, Tommy Nylander
The effects of different lipolytic enzymes on the structure of lipid liquid crystalline nano-particles (LCNP) have been investigated by cryogenic transmission electron microscopy (cryo-TEM) and synchrotron small angle X-ray diffraction (SAXD). Here we used highly structured cubic micellar (Fd3m) nanoparticles of 50/50 (wt%/wt%) soy phosphatidyl choline (SPC)/glycerol dioleate (GDO) as substrate. Two types of lipolytic enzymes were used, phospholipase A2 (PLA2) that catalysis degradation of the phospholipid component, SPC, and porcine pancreatic triacylglycerol lipase (TGL) that facilitate the hydrolysis of the diglyceride, GDO...
November 10, 2017: Chemistry and Physics of Lipids
https://www.readbyqxmd.com/read/29132296/a-fragment-based-method-for-modeling-of-protein-segments-into-cryo-em-density-maps
#13
Jochen Ismer, Alexander S Rose, Johanna K S Tiemann, Peter W Hildebrand
BACKGROUND: Single-particle analysis of electron cryo-microscopy (cryo-EM) is a key technology for elucidation of macromolecular structures. Recent technical advances in hardware and software developments significantly enhanced the resolution of cryo-EM density maps and broadened the applicability and the circle of users. To facilitate modeling of macromolecules into cryo-EM density maps, fast and easy to use methods for modeling are now demanded. RESULTS: Here we investigated and benchmarked the suitability of a classical and well established fragment-based approach for modeling of segments into cryo-EM density maps (termed FragFit)...
November 13, 2017: BMC Bioinformatics
https://www.readbyqxmd.com/read/29131938/a-crash-course-in-calcium-channels
#14
Gerald W Zamponi
Much progress has been made in understanding the molecular physiology and pharmacology of calcium channels. Recently, there have been tremendous advances in learning about calcium channel structure and function through crystallography and cryo-electron microscopy studies. Here, I will give an overview of our knowledge about calcium channels, and highlight two recent studies that give important insights into calcium channel structure.
November 13, 2017: ACS Chemical Neuroscience
https://www.readbyqxmd.com/read/29127347/unique-architecture-of-thermophilic-archaeal-virus-apbv1-and-its-genome-packaging
#15
Denis Ptchelkine, Ashley Gillum, Tomohiro Mochizuki, Soizick Lucas-Staat, Ying Liu, Mart Krupovic, Simon E V Phillips, David Prangishvili, Juha T Huiskonen
Archaeal viruses have evolved to infect hosts often thriving in extreme conditions such as high temperatures. However, there is a paucity of information on archaeal virion structures, genome packaging, and determinants of temperature resistance. The rod-shaped virus APBV1 (Aeropyrum pernix bacilliform virus 1) is among the most thermostable viruses known; it infects a hyperthermophile Aeropyrum pernix, which grows optimally at 90 °C. Here we report the structure of APBV1, determined by cryo-electron microscopy at near-atomic resolution...
November 10, 2017: Nature Communications
https://www.readbyqxmd.com/read/29120325/structural-insight-into-tpx2-stimulated-microtubule-assembly
#16
Rui Zhang, Johanna Roostalu, Thomas Surrey, Eva Nogales
During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements ('ridge' and 'wedge') in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces...
November 9, 2017: ELife
https://www.readbyqxmd.com/read/29120183/thermoresponsive-surface-grafted-gels-controlling-the-bulk-volume-change-properties-by-surface-localized-polymer-grafting-with-various-densities
#17
Ko Matsukawa, Tsukuru Masuda, Youn Soo Kim, Aya Mizutani Akimoto, Ryo Yoshida
We prepared poly(N-isopropylacrylamide-r-N-3-(aminopropyl)methacrylamide) (poly(NIPAAm-r-NAPMAm)) gels with poly NIPAAm (PNIPAAm) grafted only in the surface region (so-called thermoresponsive surface-grafted gels) with various graft densities and investigated the effect of the graft density on the bulk volume change properties, shrinking and swelling, in response to temperature changes. Initiators for atom-transfer radical polymerization (ATRP) and structurally analogous compounds were introduced at certain ratios onto the surface regions of the gels, and a subsequent activator regeneration by electron transfer ATRP of NIPAAm was conducted in aqueous media...
November 20, 2017: Langmuir: the ACS Journal of Surfaces and Colloids
https://www.readbyqxmd.com/read/29119431/observation-of-bacteriophage-ultrastructure-by-cryo-electron-microscopy
#18
Ana Cuervo, José L Carrascosa
Transmission Electron Microscopy (TEM) is an ideal method to observe and determine the structure of bacteriophages. From early studies by negative staining to the present atomic structure models derived from cryo-TEM, bacteriophage detection, classification, and structure determination has been mostly done by electron microscopy. Although embedding in metal salts has been a routine method for virus observation for many years, preservation of bacteriophages in a thin layer of fast frozen buffer has proven to be a most convenient preparation method for obtaining images using cryo-electron microscopy (cryo-EM)...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29118099/the-potential-of-cryo-electron-microscopy-for-structure-based-drug-design
#19
REVIEW
Andreas Boland, Leifu Chang, David Barford
Structure-based drug design plays a central role in therapeutic development. Until recently, protein crystallography and NMR have dominated experimental approaches to obtain structural information of biological molecules. However, in recent years rapid technical developments in single particle cryo-electron microscopy (cryo-EM) have enabled the determination to near-atomic resolution of macromolecules ranging from large multi-subunit molecular machines to proteins as small as 64 kDa. These advances have revolutionized structural biology by hugely expanding both the range of macromolecules whose structures can be determined, and by providing a description of macromolecular dynamics...
November 8, 2017: Essays in Biochemistry
https://www.readbyqxmd.com/read/29118098/x-ray-free-electron-laser-opportunities-for-drug-discovery
#20
REVIEW
Robert K Y Cheng, Rafael Abela, Michael Hennig
Past decades have shown the impact of structural information derived from complexes of drug candidates with their protein targets to facilitate the discovery of safe and effective medicines. Despite recent developments in single particle cryo-electron microscopy, X-ray crystallography has been the main method to derive structural information. The unique properties of X-ray free electron laser (XFEL) with unmet peak brilliance and beam focus allow X-ray diffraction data recording and successful structure determination from smaller and weaker diffracting crystals shortening timelines in crystal optimization...
November 8, 2017: Essays in Biochemistry
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