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 Survey of the analysis of continuous conformational variability of biological macromolecules by electron microscopy

2019 ◽  
Vol 75 (1) ◽  
pp. 19-32 ◽  
Author(s):  
C. O. S. Sorzano ◽  
A. Jiménez ◽  
J. Mota ◽  
J. L. Vilas ◽  
D. Maluenda ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Image Processing ◽  
High Resolution ◽  
Conformational Changes ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particle Analysis ◽  
Conformational Variability ◽  
Established Technique

Single-particle analysis by electron microscopy is a well established technique for analyzing the three-dimensional structures of biological macromolecules. Besides its ability to produce high-resolution structures, it also provides insights into the dynamic behavior of the structures by elucidating their conformational variability. Here, the different image-processing methods currently available to study continuous conformational changes are reviewed.

Advances in High Resolution Cryo-EM Studies: the Quartenary Structure of the 70S E.Coli Ribosome Revealed at 11.5Å Resolution.

2000 ◽  
Vol 6 (S2) ◽  
pp. 262-263
Author(s):  
P. Penczek ◽  
Ch.M.T. Spahn ◽  
R.K. Agrawal ◽  
I.S. Gabashvili ◽  
R.A. Grassucci ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Contrast Transfer Function ◽  
Multivariate Statistical ◽  
Macromolecular Assemblies ◽  
Particle Form ◽  
Common Lines

Three-dimensional cryo-electron microscopy (EM) of macromolecular assemblies with low or nonexistent symmetry in single-particle form is now recognized as a powerful, legitimate method of structure research. The progress in the resolution achieved has always been related to the introduction of new image processing methods and to improvements in understanding of the overall strategy of single particle analysis. The new techniques either were adopted from methods developed in other fields, or were specifically designed to deal with extremely low Signal-to-Noise Ratio (SNR) data in EM. In the former category we can place multivariate statistical analysis, clustering techniques, correlation techniques, and tomographic methods, in the latter alignment methods, three-dimensional (3D) projection alignment, contrast transfer function (CTF) correction methods, and common-lines based orientational search. In addition, there have been steady improvements of the quality of the data by seeking better electron microscopy conditions (usage of higher microscope voltage and of microscopes equipped with a field emission gun).

SINGLE PARTICLE ANALYSIS OF FIXED FOG DROPLETS USING SR X-RAY MICROPROBE SYSTEM

2001 ◽  
Vol 32 ◽  
pp. 873-874
Author(s):  
S. TOHNO ◽  
S. HAYAKAWA ◽  
A. NAKAMURA ◽  
A. HAMAMOTO ◽  
M. SUZUKI ◽  
...  
Keyword(s):  
Single Particle ◽  
Particle Analysis ◽  
Single Particle Analysis ◽  
X Ray
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