Project description:Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.
Project description:We target the problem of estimating the center of mass of objects in noisy two-dimensional images. We assume that the noise dominates the image, and thus many standard approaches are vulnerable to estimation errors, e.g., the direct computation of the center of mass and the geometric median which is a robust alternative to the center of mass. In this paper, we define a novel surrogate function to the center of mass. We present a mathematical and numerical analysis of our method and show that it outperforms existing methods for estimating the center of mass of an object in various realistic scenarios. As a case study, we apply our centering method to data from single-particle cryo-electron microscopy (cryo-EM), where the goal is to reconstruct the three-dimensional structure of macromolecules. We show how to apply our approach for a better translational alignment of molecule images picked from experimental data. In this way, we facilitate the succeeding steps of reconstruction and streamline the entire cryo-EM pipeline, saving computational time and supporting resolution enhancement.
Project description:We describe an implementation of maximum likelihood classification for single particle electron cryo-microscopy that is based on the FREALIGN software. Particle alignment parameters are determined by maximizing a joint likelihood that can include hierarchical priors, while classification is performed by expectation maximization of a marginal likelihood. We test the FREALIGN implementation using a simulated dataset containing computer-generated projection images of three different 70S ribosome structures, as well as a publicly available dataset of 70S ribosomes. The results show that the mixed strategy of the new FREALIGN algorithm yields performance on par with other maximum likelihood implementations, while remaining computationally efficient.
Project description:Background and objectiveThe contrast of cryo-EM images varies from one to another, primarily due to the uneven thickness of the ice layer. This contrast variation can affect the quality of 2-D class averaging, 3-D ab-initio modeling, and 3-D heterogeneity analysis. Contrast estimation is currently performed during 3-D iterative refinement. As a result, the estimates are not available at the earlier computational stages of class averaging and ab-initio modeling. This paper aims to solve the contrast estimation problem directly from the picked particle images in the ab-initio stage, without estimating the 3-D volume, image rotations, or class averages.MethodsThe key observation underlying our analysis is that the 2-D covariance matrix of the raw images is related to the covariance of the underlying clean images, the noise variance, and the contrast variability between images. We show that the contrast variability can be derived from the 2-D covariance matrix and we apply the existing Covariance Wiener Filtering (CWF) framework to estimate it. We also demonstrate a modification of CWF to estimate the contrast of individual images.ResultsOur method improves the contrast estimation by a large margin, compared to the previous CWF method. Its estimation accuracy is often comparable to that of an oracle that knows the ground truth covariance of the clean images. The more accurate contrast estimation also improves the quality of image restoration as demonstrated in both synthetic and experimental datasets.ConclusionsThis paper proposes an effective method for contrast estimation directly from noisy images without using any 3-D volume information. It enables contrast correction in the earlier stage of single particle analysis, and may improve the accuracy of downstream processing.
Project description:Three-dimensional (3D) cryoelectron microscopy reconstruction methods are uniquely able to reveal structures of many important macromolecules and macromolecular complexes. EMDataBank.org, a joint effort of the Protein Databank in Europe (PDBe), the Research Collaboratory for Structural Bioinformatics (RCSB), and the National Center for Macromolecular Imaging (NCMI), is a "one-stop shop" resource for global deposition and retrieval of cryo-EM map, model, and associated metadata. The resource unifies public access to the two major EM Structural Data archives: EM Data Bank (EMDB) and Protein Data Bank (PDB), and facilitates use of EM structural data of macromolecules and macromolecular complexes by the wider scientific community.
Project description:We present an approach to study macromolecular assemblies by detecting component proteins' characteristic high-resolution projection patterns, calculated from their known 3D structures, in single electron cryo-micrographs. Our method detects single apoferritin molecules in vitreous ice with high specificity and determines their orientation and location precisely. Simulations show that high spatial-frequency information and-in the presence of protein background-a whitening filter are essential for optimal detection, in particular for images taken far from focus. Experimentally, we could detect small viral RNA polymerase molecules, distributed randomly among binding locations, inside rotavirus particles. Based on the currently attainable image quality, we estimate a threshold for detection that is 150 kDa in ice and 300 kDa in 100 nm thick samples of dense biological material.
Project description:Single-particle analysis (SPA) in cryo-electron microscopy has become a powerful tool for determining and studying the macromolecular structure at an atomic level. However, since the SPA problem is a non-convex optimization problem with enormous search space and there is high level of noise in the input images, the existing methods may produce biased or even wrong final models. In this work, to deal with the problem, consistent constraints from the input data are explored in an embedding space, a 3D spherical surface. More specifically, the orientation of a projection image is represented by two intersection points of the normal vector and the local X-axis vector of the projection image on the unit spherical surface. To determine the orientations of the projection images, the global consistency constraints of the relative orientations of all the projection images are satisfied by two spherical embeddings which estimate the normal vectors and the local X-axis vectors of the projection images respectively. Compared to the traditional methods, the proposed method is shown to be able to rectify the initial computation errors and produce a more accurate estimation of the projection angles, which results in a better final model reconstruction from the noisy image data.
Project description:Structural heterogeneity of particles can be investigated by their three-dimensional principal components. This paper addresses the question of whether, and with what algorithm, the three-dimensional principal components can be directly recovered from cryo-EM images. The first part of the paper extends the Fourier slice theorem to covariance functions showing that the three-dimensional covariance, and hence the principal components, of a heterogeneous particle can indeed be recovered from two-dimensional cryo-EM images. The second part of the paper proposes a practical algorithm for reconstructing the principal components directly from cryo-EM images without the intermediate step of calculating covariances. This algorithm is based on maximizing the posterior likelihood using the Expectation-Maximization algorithm. The last part of the paper applies this algorithm to simulated data and to two real cryo-EM data sets: a data set of the 70S ribosome with and without Elongation Factor-G (EF-G), and a data set of the influenza virus RNA dependent RNA Polymerase (RdRP). The first principal component of the 70S ribosome data set reveals the expected conformational changes of the ribosome as the EF-G binds and unbinds. The first principal component of the RdRP data set reveals a conformational change in the two dimers of the RdRP.
Project description:Electron cryo-microscopy (cryo-EM) is increasingly being used to determine 3D structures of a broad spectrum of biological specimens from molecules to cells. Anticipating this progress in the early 2000s, an international collaboration of scientists with expertise in both cryo-EM and structure data archiving was established (EMDataResource, previously known as EMDataBank). The major goals of the collaboration have been twofold: to develop the necessary infrastructure for archiving cryo-EM-derived density maps and models, and to promote development of cryo-EM structure validation standards. We describe how cryo-EM data archiving and validation have been developed and jointly coordinated for the Electron Microscopy Data Bank and Protein Data Bank archives over the past two decades, as well as the impact of evolving technology on data standards. Just as for X-ray crystallography and nuclear magnetic resonance, engaging the scientific community via workshops and challenging activities has played a central role in developing recommendations and requirements for the cryo-EM structure data archives.
Project description:MotivationCryo-electron microscopy (cryo-EM) is a widely used technology for ultrastructure determination, which constructs the 3D structures of protein and macromolecular complex from a set of 2D micrographs. However, limited by the electron beam dose, the micrographs in cryo-EM generally suffer from the extremely low signal-to-noise ratio (SNR), which hampers the efficiency and effectiveness of downstream analysis. Especially, the noise in cryo-EM is not simple additive or multiplicative noise whose statistical characteristics are quite different from the ones in natural image, extremely shackling the performance of conventional denoising methods.ResultsHere, we introduce the Noise-Transfer2Clean (NT2C), a denoising deep neural network (DNN) for cryo-EM to enhance image contrast and restore specimen signal, whose main idea is to improve the denoising performance by correctly learning the noise distribution of cryo-EM images and transferring the statistical nature of noise into the denoiser. Especially, to cope with the complex noise model in cryo-EM, we design a contrast-guided noise and signal re-weighted algorithm to achieve clean-noisy data synthesis and data augmentation, making our method authentically achieve signal restoration based on noise's true properties. Our work verifies the feasibility of denoising based on mining the complex cryo-EM noise patterns directly from the noise patches. Comprehensive experimental results on simulated datasets and real datasets show that NT2C achieved a notable improvement in image denoising, especially in background noise removal, compared with the commonly used methods. Moreover, a case study on the real dataset demonstrates that NT2C can greatly alleviate the obstacles caused by the SNR to particle picking and simplify the identifying of particles.Availabilityand implementationThe code is available at https://github.com/Lihongjia-ict/NoiseTransfer2Clean/.Supplementary informationSupplementary data are available at Bioinformatics online.