Uncovering the structures of modular polyketide synthases

2015 ◽  
Vol 32 (3) ◽  
pp. 436-453 ◽  
Author(s):  
Kira J. Weissman
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Structural Characterization ◽  
Small Angle ◽  
Single Particle ◽  
Polyketide Synthases ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Microscopy

This review covers a breakthrough in the structural biology of the gigantic modular polyketide synthases (PKS): the structural characterization of intact modules by single-particle cryo-electron microscopy and small-angle X-ray scattering.

scholarly journals Structure of the Ebola virus nucleoprotein – RNA complex

2019 ◽  
Author(s):  
Robert N. Kirchdoerfer ◽  
Erica Ollmann Saphire ◽  
Andrew B. Ward
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Ebola Virus ◽  
Viral Proteins ◽  
Cryo Electron Microscopy ◽  
Emerging Virus ◽  
Deadly Disease ◽  
Rna Complex ◽  
Viral Nucleocapsid

AbstractEbola virus is an emerging virus capable of causing a deadly disease in humans. Replication, transcription and packaging of the viral genome is carried out by the viral nucleocapsid. The nucleocapsid is a complex of the viral nucleoprotein, RNA and several other viral proteins. The nucleoprotein NP forms large, RNA-bound, helical filaments and acts as a scaffold for additional viral proteins. The 3.1 Å single-particle cryo-electron microscopy structure of the nucleoprotein-RNA helical filament presented here resembles previous structures determined at lower resolution while providing improved molecular details of protein-protein and protein-RNA interactions. The higher resolution of the structure presented here will facilitate the design and characterization of novel and specific Ebola virus therapeutics targeting the nucleocapsid.SynopsisThe 3.1 Å single-particle cryo-electron microscopy structure of the RNA-bound, Ebola virus nucleoprotein helical filament provides molecular details of protein-protein and protein-RNA interactions.

scholarly journals 3249 Defining the Extracellular Vesicle Content of Interstitial Fluid for Blood-Free Diagnostics; Extraction Methods and Initial Characterization

2019 ◽  
Vol 3 (s1) ◽  
pp. 7-7
Author(s):  
Justin Baca Robert Taylor ◽  
Srinivasa Rao Gadam ◽  
Lauren Perez
Keyword(s):  
Electron Microscopy ◽  
Minimally Invasive ◽  
Intercellular Communication ◽  
Interstitial Fluid ◽  
Human Subjects ◽  
Extracellular Vesicle ◽  
Vis Spectroscopy ◽  
Initial Characterization

OBJECTIVES/SPECIFIC AIMS: Recent advances in microneedle technology have enabled practical, in vivo dermal interstitial fluid (ISF) sampling. These minimally-invasive techniques allow for collection of ISF without damage to adjacent tissues and do not rely on blister formation. Initial reports of extracellular vesicle (EV) isolation from dermal ISF and paired blood samples suggest that EVs may be more abundant in ISF. Analysis of ISF-derived EVs may allow for more detailed study of intercellular communication at the tissue level, particularly in acute inflammatory conditions. The objective of this study is to describe the isolation and initial characterization of interstitial fluid-derived exosomes. METHODS/STUDY POPULATION: We apply electron microscopy, nanoparticle tracking analysis (NTA), immuochemical, and sequencing methods to describe and distinguish the EV content of interstitial fluid. We include apparently healthy adult human subjects with no active skin disease. We also study immunocompetent, CD-hairless rats to demonstrate the generalizability of the methods. RESULTS/ANTICIPATED RESULTS: We successfully isolated EVs from human and rat interstitial fluid using commercially available precipitation methods. The EVs were initially characterized using UV/Vis spectroscopy, electron microscopy, and NTA. While the study is ongoing, initial results suggest that the concentration and size distribution of EVs differs significantly between blood fractions and ISF. Further immunochemical and sequencing characterization is ongoing. DISCUSSION/SIGNIFICANCE OF IMPACT: We present here the initial characterization of EVs isolated from dermal interstitial fluid. This appears to be the first report of EV characterization using ISF collection methods that do not perturb adjacent tissues (such as with blister or microdialysis methods). The present study lays a foundation for further examination of ISF-derived EVs in acute inflammatory disease such as cellulitis or infectious neuritis. This may enable minimally invasive diagnostics and new research tools to understand intercellular communication in living organisms with increased spatial and temporal resolution.

scholarly journals Structural characterization of NDH-1 complexes of Thermosynechococcus elongatus by single particle electron microscopy

2006 ◽  
Vol 1757 (11) ◽  
pp. 1469-1475 ◽  
Author(s):  
Ana A. Arteni ◽  
Pengpeng Zhang ◽  
Natalia Battchikova ◽  
Teruo Ogawa ◽  
Eva-Mari Aro ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Structural Characterization ◽  
Single Particle ◽  
Single Particle Electron Microscopy

scholarly journals Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids

2016 ◽  
Author(s):  
Joost Snijder ◽  
Andrew J. Borst ◽  
Annie Dosey ◽  
Alexandra C. Walls ◽  
Anika Burrell ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Particle Density ◽  
Protein Complexes ◽  
Cost Effective ◽  
Carbon Support ◽  
Cryo Electron Microscopy ◽  
Sample Application ◽  
Single Field

Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain a dense distribution of particles within a single field of view remains a major bottleneck for the success of such experiments. Here, we describe a simple and cost-effective method to increase the density of frozen-hydrated particles on grids with holey carbon support films. It relies on performing multiple rounds of sample application and blotting prior to plunge freezing in liquid ethane. We show that this approach is generally applicable and significantly increases particle density for a range of samples, such as small protein complexes, viruses and filamentous assemblies. The method is versatile, easy to implement, minimizes sample requirements and can enable characterization of samples that would otherwise resist structural studies using single particle cryoEM.

Localization of immunolabels by electron microscopy and image averaging

1983 ◽  
Vol 41 ◽  
pp. 282-283
Author(s):  
J. Frank ◽  
P.-Y. Sizaret ◽  
A. Verschoor ◽  
J. Lamy
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Attachment Site ◽  
Uranyl Acetate ◽  
Limulus Polyphemus ◽  
Resolution Limit ◽  
Electron Microscopic ◽  
Image Averaging ◽  
Top View ◽  
Better Than

The accuracy with which the attachment site of immunolabels bound to macromolecules may be localized in electron microscopic images can be considerably improved by using single particle averaging. The example studied in this work showed that the accuracy may be better than the resolution limit imposed by negative staining (∽2nm).The structure used for this demonstration was a halfmolecule of Limulus polyphemus (LP) hemocyanin, consisting of 24 subunits grouped into four hexamers. The top view of this structure was previously studied by image averaging and correspondence analysis. It was found to vary according to the flip or flop position of the molecule, and to the stain imbalance between diagonally opposed hexamers (“rocking effect”). These findings have recently been incorporated into a model of the full 8 × 6 molecule.LP hemocyanin contains eight different polypeptides, and antibodies specific for one, LP II, were used. Uranyl acetate was used as stain. A total of 58 molecule images (29 unlabelled, 29 labelled with antl-LPII Fab) showing the top view were digitized in the microdensitometer with a sampling distance of 50μ corresponding to 6.25nm.

Characterization of Tamm-Horsfall Urinary Glycoprotein

1973 ◽  
Vol 31 ◽  
pp. 420-421
Author(s):  
John P. Robinson ◽  
J. David Puett
Keyword(s):  
Electron Microscopy ◽  
Circular Dichroism ◽  
Secondary Structure ◽  
Quaternary Structure ◽  
Normal Adult ◽  
Morphological Properties ◽  
Adult Males ◽  
Circular Dichroïsm ◽  
Urinary Glycoprotein

Much work has been reported on the chemical, physical and morphological properties of urinary Tamm-Horsfall glycoprotein (THG). Although it was once reported that cystic fibrotic (CF) individuals had a defective THG, more recent data indicate that THG and CF-THG are similar if not identical.No studies on the conformational aspects have been reported on this glycoprotein using circular dichroism (CD). We examined the secondary structure of THG and derivatives under various conditions and have correlated these results with quaternary structure using electron microscopy.THG was prepared from normal adult males and CF-THG from a 16-year old CF female by the method of Tamm and Horsfall. CF female by the method of Tamm and Horsfall.

Electron Microscopic Detection and Characterization of Nonhuman Primate Enteric Viruses

1982 ◽  
Vol 40 ◽  
pp. 306-307
Author(s):  
G. C. Smith ◽  
R. L. Heberling ◽  
S. S. Kalter
Keyword(s):  
Electron Microscopy ◽  
Animal Model ◽  
Nonhuman Primate ◽  
Clinical Condition ◽  
Intestinal Tract ◽  
Enteric Viruses ◽  
Electron Microscopic ◽  
Microscopic Detection

A number of viral agents are recognized as and suspected of causing the clinical condition “gastroenteritis.” In our attempts to establish an animal model for studies of this entity, we have been examining the nonhuman primate to ascertain what viruses may be found in the intestinal tract of “normal” animals as well as animals with diarrhea. Several virus types including coronavirus, adenovirus, herpesvirus, and picornavirus (Table I) were detected in our colony; however, rotavirus, astrovirus, and calicivirus have not yet been observed. Fecal specimens were prepared for electron microscopy by procedures reported previously.

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