Methods are presented that detect three types of aberrations in single-particle cryo-EM data sets: symmetrical and antisymmetrical optical aberrations and magnification anisotropy. Because these ...methods only depend on the availability of a preliminary 3D reconstruction from the data, they can be used to correct for these aberrations for any given cryo-EM data set,
. Using five publicly available data sets, it is shown that considering these aberrations improves the resolution of the 3D reconstruction when these effects are present. The methods are implemented in version 3.1 of the open-source software package
.
Helical reconstruction in RELION is increasingly being used to determine the atomic structures of amyloid filaments from electron cryo‐microscopy (cryo‐EM) images. However, because the energy ...landscape of amyloid refinements is typically fraught with local optima, amyloid structure determination is often difficult. This paper aims to help RELION users in this process. It discusses aspects of helical reconstruction that are particularly relevant to amyloids, it illustrates the problem of local optima in refinement and how to detect them, and it introduces a new method to calculate 3D initial models from reference‐free 2D class averages. By providing starting models that are closer to the global optimum, this method makes amyloid structure determination easier. All methods described are open‐source and distributed within RELION‐3.1. Their use is illustrated using a publicly available data set on tau filaments from the brain of an individual with Alzheimer's disease.
Amyloid structure determination in RELION is increasingly popular, but may suffer from refinements becoming stuck in local minima. This paper describes a new method for initial model generation that may alleviate this problem.
A new method to estimate the trajectories of particle motion and the amount of cumulative beam damage in electron cryo-microscopy (cryo-EM) single-particle analysis is presented. The motion within ...the sample is modelled through the use of Gaussian process regression. This allows a prior likelihood that favours spatially and temporally smooth motion to be associated with each hypothetical set of particle trajectories without imposing hard constraints. This formulation enables the
likelihood of a set of particle trajectories to be expressed as a product of that prior likelihood and an observation likelihood given by the data, and this
likelihood to then be maximized. Since the smoothness prior requires three parameters that describe the statistics of the observed motion, an efficient stochastic method to estimate these parameters is also proposed. Finally, a practical algorithm is proposed that estimates the average amount of cumulative radiation damage as a function of radiation dose and spatial frequency, and then fits relative
factors to that damage in a robust way. The method is evaluated on three publicly available data sets, and its usefulness is illustrated by comparison with state-of-the-art methods and previously published results. The new method has been implemented as Bayesian polishing in
-3, where it replaces the existing particle-polishing method, as it outperforms the latter in all tests conducted.
Knowledge of the three-dimensional structures of proteins and other biological macromolecules often aids understanding of how they perform complicated tasks in the cell. Because many such tasks ...involve the cleavage or formation of chemical bonds, structural characterization at the atomic level is most useful. Developments in the electron microscopy of frozen hydrated samples (cryo-electron microscopy) are providing unprecedented opportunities for the structural characterization of biological macromolecules. This is resulting in a wave of information about processes in the cell that were impossible to characterize with existing techniques in structural biology.
RELION, for REgularized LIkelihood OptimizatioN, is an open-source computer program for the refinement of macromolecular structures by single-particle analysis of electron cryo-microscopy (cryo-EM) ...data. Whereas alternative approaches often rely on user expertise for the tuning of parameters, RELION uses a Bayesian approach to infer parameters of a statistical model from the data. This paper describes developments that reduce the computational costs of the underlying maximum a posteriori (MAP) algorithm, as well as statistical considerations that yield new insights into the accuracy with which the relative orientations of individual particles may be determined. A so-called gold-standard Fourier shell correlation (FSC) procedure to prevent overfitting is also described. The resulting implementation yields high-quality reconstructions and reliable resolution estimates with minimal user intervention and at acceptable computational costs.
Three-dimensional (3D) structure determination by single-particle analysis of cryo-electron microscopy (cryo-EM) images requires many parameters to be determined from extremely noisy data. This makes ...the method prone to overfitting, that is, when structures describe noise rather than signal, in particular near their resolution limit where noise levels are highest. Cryo-EM structures are typically filtered using ad hoc procedures to prevent overfitting, but the tuning of arbitrary parameters may lead to subjectivity in the results. I describe a Bayesian interpretation of cryo-EM structure determination, where smoothness in the reconstructed density is imposed through a Gaussian prior in the Fourier domain. The statistical framework dictates how data and prior knowledge should be combined, so that the optimal 3D linear filter is obtained without the need for arbitrariness and objective resolution estimates may be obtained. Application to experimental data indicates that the statistical approach yields more reliable structures than existing methods and is capable of detecting smaller classes in data sets that contain multiple different structures.
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► Limiting overfitting in cryo-EM reconstruction requires user expertise. ► A Bayesian approach may limit overfitting without arbitrary decisions, providing novel insights in the Wiener filter for 3D reconstruction, yielding better structures and improved classifications, and contributing to increase the objectivity in cryo-EM reconstruction.
The highly divergent ribosomes of human mitochondria (mitoribosomes) synthesize 13 essential proteins of oxidative phosphorylation complexes. We have determined the structure of the intact ...mitoribosome to 3.5 angstrom resolution by means of single-particle electron cryogenic microscopy. It reveals 80 extensively interconnected proteins, 36 of which are specific to mitochondria, and three ribosomal RNA molecules. The head domain of the small subunit, particularly the messenger (mRNA) channel, is highly remodeled. Many intersubunit bridges are specific to the mitoribosome, which adopts conformations involving ratcheting or rolling of the small subunit that are distinct from those seen in bacteria or eukaryotes. An intrinsic guanosine triphosphatase mediates a contact between the head and central protuberance. The structure provides a reference for analysis of mutations that cause severe pathologies and for future drug design.
3D cryo-electron microscopy (cryo-EM) is an expanding structural biology technique that has recently undergone a quantum leap progression in its achievable resolution and its applicability to the ...study of challenging biological systems. Because crystallization is not required, only small amounts of sample are needed, and because images can be classified in a computer, the technique has the potential to deal with compositional and conformational mixtures. Therefore, cryo-EM can be used to investigate complete and fully functional macromolecular complexes in different functional states, providing a richness of biological insight. In this review, we underlie some of the principles behind the cryo-EM methodology of single particle analysis and discuss some recent results of its application to challenging systems of paramount biological importance. We place special emphasis on new methodological developments that are leading to an explosion of new studies, many of which are reaching resolutions that could only be dreamed of just a couple of years ago.
Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial ...diseases and are often the unintended targets of various clinically useful antibiotics. Using single-particle cryogenic electron microscopy, we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance, including recruitment of mitochondrial valine transfer RNA (tRNAVal) to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs.
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