Determining the structure of biological macromolecules by transmission electron microscopy, single particle analysis and 3D reconstruction

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.

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

Crystals ◽

10.3390/cryst10070580 ◽

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.

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Structural analysis of DNA wrapping in bacterial transcription initiation complex by transmission electron microscopy and single particle analysis

10.11606/d.76.2018.tde-19102018-090529 ◽

2018 ◽

Author(s):

Alfredo Jose Florez Ariza

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Structural Analysis ◽

Transcription Initiation ◽

Particle Analysis ◽

Single Particle Analysis ◽

Initiation Complex ◽

Bacterial Transcription ◽

Transmission Electron ◽

Transcription Initiation Complex

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Snapshots of single particles from single cells using electron microscopy

10.1101/435222 ◽

2018 ◽

Cited By ~ 1

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.

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