Technical approaches of single particle analysis following electron microscopy to pre-screening biological candidates targeted on high resolution studies

2010 ◽  
Vol 1 (1) ◽  
pp. 66-70
Author(s):  
Hyun Suk Jung
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Single Particle ◽  
Particle Analysis ◽  
Single Particle Analysis

scholarly journals Electron microscopy snapshots of single particles from single cells

2018 ◽  
Vol 294 (5) ◽  
pp. 1602-1608 ◽  
Author(s):  
Xiunan Yi ◽  
Eric J. Verbeke ◽  
Yiran Chang ◽  
Daniel J. Dickinson ◽  
David W. Taylor
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Protein Complexes ◽  
Signal To Noise Ratio ◽  
Single Cells ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particle Analysis ◽  
Whole Cells ◽  
Potential Applications

Cryo-electron microscopy (cryo-EM) has become an indispensable tool for structural studies of biological macromolecules. Two additional predominant methods are available for studying the architectures of multiprotein complexes: 1) single-particle analysis of purified samples and 2) tomography of whole cells or cell sections. The former can produce high-resolution structures but is limited to highly purified samples, whereas the latter can capture proteins in their native state but has a low signal-to-noise ratio and yields lower-resolution structures. Here, we present a simple, adaptable method combining microfluidic single-cell extraction with single-particle analysis by EM to characterize protein complexes from individual Caenorhabditis elegans embryos. Using this approach, we uncover 3D structures of ribosomes directly from single embryo extracts. Moreover, we investigated structural dynamics during development by counting the number of ribosomes per polysome in early and late embryos. This approach has significant potential applications for counting protein complexes and studying protein architectures from single cells in developmental, evolutionary, and disease contexts.

scholarly journals Single particle analysis of clamp loader by electron microscopy

1999 ◽  
Vol 39 (supplement) ◽  
pp. S111
Author(s):  
K. Mayanagi ◽  
T. Miyata ◽  
K. Morikawa ◽  
I. Cann ◽  
S. Ishino ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Particle Analysis ◽  
Single Particle Analysis ◽  
Clamp Loader

scholarly journals The Resolution in X-ray Crystallography and Single-Particle Cryogenic Electron Microscopy

Crystals ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 580
Author(s):  
Victor R.A. Dubach ◽  
Albert Guskov
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particle Analysis ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Fourier Transform

X-ray crystallography and single-particle analysis cryogenic electron microscopy are essential techniques for uncovering the three-dimensional structures of biological macromolecules. Both techniques rely on the Fourier transform to calculate experimental maps. However, one of the crucial parameters, resolution, is rather broadly defined. Here, the methods to determine the resolution in X-ray crystallography and single-particle analysis are summarized. In X-ray crystallography, it is becoming increasingly more common to include reflections discarded previously by traditionally used standards, allowing for the inclusion of incomplete and anisotropic reflections into the refinement process. In general, the resolution is the smallest lattice spacing given by Bragg’s law for a particular set of X-ray diffraction intensities; however, typically the resolution is truncated by the user during the data processing based on certain parameters and later it is used during refinement. However, at which resolution to perform such a truncation is not always clear and this makes it very confusing for the novices entering the structural biology field. Furthermore, it is argued that the effective resolution should be also reported as it is a more descriptive measure accounting for anisotropy and incompleteness of the data. In single particle cryo-EM, the situation is not much better, as multiple ways exist to determine the resolution, such as Fourier shell correlation, spectral signal-to-noise ratio and the Fourier neighbor correlation. The most widely accepted is the Fourier shell correlation using a threshold of 0.143 to define the resolution (so-called “gold-standard”), although it is still debated whether this is the correct threshold. Besides, the resolution obtained from the Fourier shell correlation is an estimate of varying resolution across the density map. In reality, the interpretability of the map is more important than the numerical value of the resolution.

scholarly journals Electron microscopy and functional analysis of recombinant innexin gap junctions

2014 ◽  
Vol 70 (a1) ◽  
pp. C851-C851
Author(s):  
Atsunori Oshima ◽  
Tomohiro Matsuzawa ◽  
Kazuyoshi Murata ◽  
Kouki Nishikawa ◽  
Yoshinori Fujiyoshi
Keyword(s):  
Electron Microscopy ◽  
Gap Junctions ◽  
Gap Junction ◽  
Single Particle ◽  
Particle Analysis ◽  
Single Particle Analysis ◽  
Dye Transfer ◽  
Gap Junction Channels ◽  
Class Average ◽  
Detergent Micelles

Innexin is a molecular component of invertebrate gap junctions, which have an important role in neural and muscular electrical activity in invertebrates. Although the structure of vertebrate connexin26 was revealed by X-ray crystallography [1], the structure of innexin channels remains poorly understood. To study the structure of innexin gap junction channels, we expressed and purified Caenorhabditis elegans innexin-6 (INX-6) gap junction channels, and characterized their molecular dimensions and channel permeability using electron microscopy (EM) and a fluorescent dye transfer assay, respectively [2]. Negative-staining and thin-section EM of isolated INX-6 gap junction plaques revealed a loosely packed hexagonal lattice. We performed single particle analysis of purified INX-6 channels with negative-staining and cryo EM. Based on the negative-stain EM images, the class average of the junction form had a longitudinal height of 220 Å, a channel diameter of 110 Å in the absence of detergent micelles, and an extracellular gap space of 60 Å, whereas the class average of the hemichannels had diameters of up to 140 Å in the presence of detergent micelles. Cryo EM images revealed rotational peaks that could be related to the INX-6 subunits. Structural analysis of the reconstituted INX-6 channels with single particle analysis and electron tomography suggested that the oligomeric number of the INX-6 channel was distinct from that of the dodecameric connexin channel. Dye transfer experiments indicated that the INX-6-GFP-His channels were permeable to 3-kDa and 10-kDa dextran-conjugated tracers. These findings indicate that INX-6 channels have a characteristic oligomer component that differs from that in connexin gap junction channels.

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