scholarly journals Quantitative analysis of 3D alignment quality: its impact on soft-validation, particle pruning and homogeneity analysis

2016 ◽  
Author(s):  
J. Vargas ◽  
R. Melero ◽  
J. Gómez-Blanco ◽  
J. M. Carazo ◽  
C. O. S. Sorzano
Keyword(s):  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particles ◽  
Homogeneity Analysis ◽  
Cryo Electron Microscopy ◽  
Alignment Process ◽  
Projection Images ◽  
Map Resolution

AbstractSingle Particle Analysis using cryo-electron microscopy is a structural biology technique to capture the three-dimensional conformation of biological macromolecules. The projection images used to construct the 3D density map are characterized by a very low signal-to-noise ratio to minimize radiation damage in the samples. As a consequence, the 3D alignment process is a challenging and error prone task and this job usually determines the success or failure of the macromolecule reconstruction. In this work, we present a soft-alignment validation approach, which can quantify the alignment precision and accuracy as well as the data homogeneity of the single particles when they are confronted with the resultant reconstructed 3DEM map. We have also applied this method to data homogeneity analysis and particle pruning, improving the data quality and as a consequence the final map resolution.

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 Visualization of biological macromolecules at near-atomic resolution: cryo-electron microscopy comes of age

2019 ◽  
Vol 75 (1) ◽  
pp. 3-11 ◽  
Author(s):  
Alok K. Mitra
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Atomic Resolution ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particles ◽  
Cryo Electron Microscopy ◽  
Electron Microscopes ◽  
Resolution Single ◽  
Resolution Structure

Structural biology is going through a revolution as a result of transformational advances in the field of cryo-electron microscopy (cryo-EM) driven by the development of direct electron detectors and ultrastable electron microscopes. High-resolution cryo-EM images of isolated biomolecules (single particles) suspended in a thin layer of vitrified buffer are subjected to powerful image-processing algorithms, enabling near-atomic resolution structures to be determined in unprecedented numbers. Prior to these advances, electron crystallography of two-dimensional crystals and helical assemblies of proteins had established the feasibility of atomic resolution structure determination using cryo-EM. Atomic resolution single-particle analysis, without the need for crystals, now promises to resolve problems in structural biology that were intractable just a few years ago.

scholarly journals Application of Symmetry Adapted Function Method for Three-Dimensional Reconstruction of Octahedral Biological Macromolecules

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Songjun Zeng ◽  
Hongrong Liu ◽  
Qibin Yang
Keyword(s):  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Biological Macromolecules ◽  
Single Particles ◽  
Dimensional Reconstruction ◽  
Standard Models ◽  
Relative Errors ◽  
High Level ◽  
Influence Of Noise ◽  
Good Agreement

A method for three-dimensional (3D) reconstruction of macromolecule assembles, that is, octahedral symmetrical adapted functions (OSAFs) method, was introduced in this paper and a series of formulations for reconstruction by OSAF method were derived. To verify the feasibility and advantages of the method, two octahedral symmetrical macromolecules, that is, heat shock proteinDegp24and the Red-cell L Ferritin, were utilized as examples to implement reconstruction by the OSAF method. The schedule for simulation was designed as follows: 2000 random orientated projections of single particles with predefined Euler angles and centers of origins were generated, then different levels of noises that is signal-to-noise ratio (S/N)=0.1,0.5, and 0.8 were added. The structures reconstructed by the OSAF method were in good agreement with the standard models and the relative errors of the structures reconstructed by the OSAF method to standard structures were very little even for high level noise. The facts mentioned above account for that the OSAF method is feasible and efficient approach to reconstruct structures of macromolecules and have ability to suppress the influence of noise.

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 A national facility for biological cryo-electron microscopy

2015 ◽  
Vol 71 (1) ◽  
pp. 127-135 ◽  
Author(s):  
Helen R. Saibil ◽  
Kay Grünewald ◽  
David I. Stuart
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Dimensional Electron ◽  
Cryo Electron Microscopy ◽  
Cellular Context ◽  
Molecular Machinery ◽  
User Friendly ◽  
Integrated Structural Biology ◽  
Expert Support

Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided of the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback.

Refinement of cryo-EM structures using scattering factors of charged atoms

2016 ◽  
Vol 49 (5) ◽  
pp. 1517-1523 ◽  
Author(s):  
Koji Yonekura ◽  
Saori Maki-Yonekura
Keyword(s):  
Structure Refinement ◽  
Three Dimensional ◽  
Electron Diffraction Data ◽  
Particle Analysis ◽  
Accurate Analysis ◽  
Atomic Models ◽  
Charged Amino Acids ◽  
Cryo Electron Microscopy ◽  
Partial Charges ◽  
Suitable Treatment

This paper reports a suitable treatment of electron scattering factors of charged atoms for refinement of atomic models against cryo-electron microscopy (cryo-EM) maps. The ScatCurve package developed here supports various curve models for parameterization of scattering factors and the parameter tables can be implemented in major refinement programs in structural biology. Partial charge values of charged amino acids in crystal structures were changed in small steps for refinement of the atomic models against electron diffraction data from three-dimensional crystals. By exploring a range of partial charges, the authors found the electrostatic setting that produces atomic models with improved statistics and better reflects experimental data. Structure refinement for single-particle analysis also benefits from the more accurate analysis and the programs could find wide use for model refinement against cryo-EM maps.

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 A WAVELET BASED ALTERNATIVE ITERATION METHOD FOR THE ORIENTATION REFINEMENT OF CRYO-ELECTRON MICROSCOPY 3D RECONSTRUCTION

2015 ◽  
Vol 20 (3) ◽  
pp. 396-408 ◽  
Author(s):  
Zhucui Jing ◽  
Ming Li
Keyword(s):  
Electron Microscopy ◽  
Iteration Method ◽  
Three Dimensional ◽  
Particle Method ◽  
Biological Macromolecules ◽  
Orthonormal Bases ◽  
Cryo Electron Microscopy ◽  
Marquardt Algorithm ◽  
Density Map ◽  
Particle Images

Cryo-electron microscopy (cryo-EM) single particle method (SPM) reconstructs the three-dimensional (3D) density map of biological macromolecules using 2D particle images with estimated orientations. The estimated orientations have errors which result in the decrease in resolution of the reconstructed map. We propose a wavelet orthonormal bases based iteration method by refining alternatively the orientations and the map using Levenberg–Marquardt algorithm and soft-thresholding, respectively. The convergence analysis of the proposed algorithm is provided and numerical experiments for simulated particle images show its good performance.

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