scholarly journals Three dimensional particle averaging for structural imaging of macromolecular complexes by localization microscopy

2019 ◽  
Author(s):  
Hamidreza Heydarian ◽  
Adrian Przybylski ◽  
Florian Schueder ◽  
Ralf Jungmann ◽  
Ben van Werkhoven ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Dna Origami ◽  
Particle Analysis ◽  
Nuclear Pore ◽  
Single Particle Analysis ◽  
Macromolecular Complexes ◽  
Localization Microscopy ◽  
Structural Template ◽  
Microscopy Techniques

AbstractWe present an approach for 3D particle fusion in localization microscopy which dramatically increases signal-to-noise ratio and resolution in single particle analysis. Our method does not require a structural template, and properly handles anisotropic localization uncertainties. We demonstrate 3D particle reconstructions of the Nup107 subcomplex of the nuclear pore complex (NPC), cross-validated using multiple localization microscopy techniques, as well as two-color 3D reconstructions of the NPC, and reconstructions of DNA-origami tetrahedrons.

scholarly journals 3D particle averaging and detection of macromolecular symmetry in localization microscopy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hamidreza Heydarian ◽  
Maarten Joosten ◽  
Adrian Przybylski ◽  
Florian Schueder ◽  
Ralf Jungmann ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Fluorescent Labeling ◽  
Dna Origami ◽  
Particle Analysis ◽  
Nuclear Pore ◽  
Localization Microscopy ◽  
Structural Template ◽  
Microscopy Techniques ◽  
Single Molecule Localization Microscopy

AbstractSingle molecule localization microscopy offers in principle resolution down to the molecular level, but in practice this is limited primarily by incomplete fluorescent labeling of the structure. This missing information can be completed by merging information from many structurally identical particles. In this work, we present an approach for 3D single particle analysis in localization microscopy which hugely increases signal-to-noise ratio and resolution and enables determining the symmetry groups of macromolecular complexes. Our method does not require a structural template, and handles anisotropic localization uncertainties. We demonstrate 3D reconstructions of DNA-origami tetrahedrons, Nup96 and Nup107 subcomplexes of the nuclear pore complex acquired using multiple single molecule localization microscopy techniques, with their structural symmetry deducted from the data.

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 Snapshots of single particles from single cells using electron microscopy

2018 ◽  
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 ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particle Analysis ◽  
Whole Cells

AbstractCryo-electron microscopy has become an indispensable tool for structural studies of biological macromolecules. There are two predominant methods for studying the architectures of multi-protein 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, while the latter can capture proteins in their native state but is hindered by a low signal-to-noise ratio and results in lower-resolution structures. Here, we present a method combining microfluidic single cell extraction with single particle analysis by electron microscopy to characterize protein complexes from individual C. elegans embryos. Using this approach, we uncover three-dimensional structures of ribosomes directly from single embryo extracts. In addition, we investigate structural dynamics during development by counting the number of ribosomes per polysome in early and late embyros. 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 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 Three Dimensional Reconstruction of CFTR Chloride Channel Using Single Particle Analysis

2009 ◽  
Vol 96 (3) ◽  
pp. 468a
Author(s):  
Kazuhiro Mio ◽  
Toshihiko Ogura ◽  
Muneyo Mio ◽  
Hiroyasu Shimizu ◽  
Tzyh-Chang Hwang ◽  
...  
Keyword(s):  
Single Particle ◽  
Chloride Channel ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Single Particle Analysis ◽  
Three Dimensional Reconstruction ◽  
Dimensional Reconstruction
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