RESUMO
Single Particle analysis (SPA) aims to determine the three-dimensional structure of proteins and macromolecular complexes. The current state of the art has allowed us to achieve near-atomic and even atomic resolutions. To obtain high-resolution structures, a set of well-defined image processing steps is required. A critical one is the estimation of the Contrast Transfer Function (CTF), which considers the sample defocus and aberrations of the microscope. Defocus is usually globally estimated; in this case, it is the same for all the particles in each micrograph. But proteins are ice-embedded at different heights, suggesting that defocus should be measured in a local (per particle) manner. There are four state-of-the-art programs to estimate local defocus (Gctf, Relion, CryoSPARC, and Xmipp). In this work, we have compared the results of these software packages to check whether the resolution improves. We have used the Scipion framework and developed a specific program to analyze local defocus. The results produced by different programs do not show a clear consensus using the current test datasets in this study.
Assuntos
Processamento de Imagem Assistida por Computador , Imagem Individual de Molécula , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Substâncias Macromoleculares , Software , AlgoritmosRESUMO
Single particle analysis (SPA) in cryo-electron microscopy (cryo-EM) is highly used to obtain the near-atomic structure of biological macromolecules. The current methods allow users to produce high-resolution maps from many samples. However, there are still challenging cases that require extra processing to obtain high resolution. This is the case when the macromolecule of the sample is composed of different components and we want to focus just on one of them. For example, if the macromolecule is composed of several flexible subunits and we are interested in a specific one, if it is embedded in a viral capsid environment, or if it has additional components to stabilize it, such as nanodiscs. The signal from these components, which in principle we are not interested in, can be removed from the particles using a projection subtraction method. Currently, there are two projection subtraction methods used in practice and both have some limitations. In fact, after evaluating their results, we consider that the problem is still open to new solutions, as they do not fully remove the signal of the components that are not of interest. Our aim is to develop a new and more precise projection subtraction method, improving the performance of state-of-the-art methods. We tested our algorithm with data from public databases and an in-house data set. In this work, we show that the performance of our algorithm improves the results obtained by others, including the localization of small ligands, such as drugs, whose binding location is unknown a priori.
Assuntos
Algoritmos , Imagem Individual de Molécula , Microscopia Crioeletrônica/métodos , Substâncias Macromoleculares/químicaRESUMO
Image processing in cryogenic electron tomography (cryoET) is currently at a similar state as Single Particle Analysis (SPA) in cryogenic electron microscopy (cryoEM) was a few years ago. Its data processing workflows are far from being well defined and the user experience is still not smooth. Moreover, file formats of different software packages and their associated metadata are not standardized, mainly since different packages are developed by different groups, focusing on different steps of the data processing pipeline. The Scipion framework, originally developed for SPA (de la Rosa-Trevín et al., 2016), has a generic python workflow engine that gives it the versatility to be extended to other fields, as demonstrated for model building (Martínez et al., 2020). In this article, we provide an extension of Scipion based on a set of tomography plugins (referred to as ScipionTomo hereafter), with a similar purpose: to allow users to be focused on the data processing and analysis instead of having to deal with multiple software installation issues and the inconvenience of switching from one to another, converting metadata files, managing possible incompatibilities, scripting (writing a simple program in a language that the computer must convert to machine language each time the program is run), etcetera. Additionally, having all the software available in an integrated platform allows comparing the results of different algorithms trying to solve the same problem. In this way, the commonalities and differences between estimated parameters shed light on which results can be more trusted than others. ScipionTomo is developed by a collaborative multidisciplinary team composed of Scipion team engineers, structural biologists, and in some cases, the developers whose software packages have been integrated. It is open to anyone in the field willing to contribute to this project. The result is a framework extension that combines the acquired knowledge of Scipion developers in close collaboration with third-party developers, and the on-demand design of functionalities requested by beta testers applying this solution to actual biological problems.
Assuntos
Tomografia com Microscopia Eletrônica , Software , Algoritmos , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Reprodutibilidade dos TestesRESUMO
The number of maps deposited in public databases (Electron Microscopy Data Bank, EMDB) determined by cryo-electron microscopy has quickly grown in recent years. With this rapid growth, it is critical to guarantee their quality. So far, map validation has primarily focused on the agreement between maps and models. From the image processing perspective, the validation has been mostly restricted to using two half-maps and the measurement of their internal consistency. In this article, we suggest that map validation can be taken much further from the point of view of image processing if 2D classes, particles, angles, coordinates, defoci, and micrographs are also provided. We present a progressive validation scheme that qualifies a result validation status from 0 to 5 and offers three optional qualifiers (A, W, and O) that can be added. The simplest validation state is 0, while the most complete would be 5AWO. This scheme has been implemented in a website https://biocomp.cnb.csic.es/EMValidationService/ to which reconstructed maps and their ESI can be uploaded.
Assuntos
Processamento de Imagem Assistida por Computador , Microscopia Crioeletrônica/métodos , Microscopia EletrônicaRESUMO
Electron cryomicroscopy (cryo-EM) has emerged as a powerful structural biology instrument to solve near-atomic three-dimensional structures. Despite the fast growth in the number of density maps generated from cryo-EM data, comparison tools among these reconstructions are still lacking. Current proposals to compare cryo-EM data derived volumes perform map subtraction based on adjustment of each volume grey level to the same scale. We present here a more sophisticated way of adjusting the volumes before comparing, which implies adjustment of grey level scale and spectrum energy, but keeping phases intact inside a mask and imposing the results to be strictly positive. The adjustment that we propose leaves the volumes in the same numeric frame, allowing to perform operations among the adjusted volumes in a more reliable way. This adjustment can be a preliminary step for several applications such as comparison through subtraction, map sharpening, or combination of volumes through a consensus that selects the best resolved parts of each input map. Our development might also be used as a sharpening method using an atomic model as a reference. We illustrate the applicability of this algorithm with the reconstructions derived of several experimental examples. This algorithm is implemented in Xmipp software package and its applications are user-friendly accessible through the cryo-EM image processing framework Scipion.
Assuntos
Algoritmos , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Substâncias Macromoleculares/ultraestrutura , Capsídeo/química , Capsídeo/ultraestrutura , Vírus da Hepatite B/ultraestrutura , Substâncias Macromoleculares/química , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Reprodutibilidade dos Testes , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/ultraestruturaRESUMO
The presence of preferred orientations in single particle analysis (SPA) by cryo-Electron Microscopy (cryoEM) is currently one of the hurdles preventing many structural analyses from yielding high-resolution structures. Although the existence of preferred orientations is mostly related to the grid preparation, in this technical note, we show that some image processing algorithms used for angular assignment and three-dimensional (3D) reconstruction are more robust than others to these detrimental conditions. We exemplify this argument with three different data sets in which the presence of preferred orientations hindered achieving a 3D reconstruction without artifacts or, even worse, a 3D reconstruction could never be achieved.
Assuntos
Microscopia Crioeletrônica/métodos , Imagem Individual de Molécula/métodos , Algoritmos , Artefatos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodosRESUMO
Advances in cryo-electron microscopy (cryo-EM) have made it possible to obtain structures of large biological macromolecules at near-atomic resolution. This "resolution revolution" has encouraged the use and development of modeling tools able to produce high-quality atomic models from cryo-EM density maps. Unfortunately, many practical problems appear when combining different packages in the same processing workflow, which make difficult the use of these tools by non-experts and, therefore, reduce their utility. We present here a major extension of the image processing framework Scipion that provides inter-package integration in the model building area and full tracking of the complete workflow, from image processing to structure validation.
Assuntos
Processamento de Imagem Assistida por Computador , Software , Microscopia Crioeletrônica , Fluxo de TrabalhoRESUMO
The introduction of Direct Electron Detector (DED) videos in the Electron Microscope field has boosted Single Particle Analysis to a point in which it is currently considered to be a key technique in Structural Biology. In this article we introduce an approach to estimate the DED camera gain at each pixel from the movies themselves. This gain is needed to have the set of recorded frames into a coherent gray level range, homogeneous over the whole image. The algorithm does not need any other input than the DED movie itself, being capable of providing an estimate of the camera gain image, helping to identify dead pixels and cases of incorrectly calibrated cameras. We propose the algorithm to be used either to validate the experimentally acquired gain image (for instance, to follow its possible change over time) or to verify that there is no residual gain image after experimentally correcting for the camera gain. We show results for a number of DED camera models currently in use (DE, Falcon II, Falcon 3, and K2).
Assuntos
Microscopia Eletrônica/métodos , Algoritmos , Microscopia Crioeletrônica , Processamento de Imagem Assistida por Computador , FotografaçãoRESUMO
Cryo-electron microscopy (cryoEM) has become a well established technique to elucidate the 3D structures of biological macromolecules. Projection images from thousands of macromolecules that are assumed to be structurally identical are combined into a single 3D map representing the Coulomb potential of the macromolecule under study. This article discusses possible caveats along the image-processing path and how to avoid them to obtain a reliable 3D structure. Some of these problems are very well known in the community. These may be referred to as sample-related (such as specimen denaturation at interfaces or non-uniform projection geometry leading to underrepresented projection directions). The rest are related to the algorithms used. While some have been discussed in depth in the literature, such as the use of an incorrect initial volume, others have received much less attention. However, they are fundamental in any data-analysis approach. Chiefly among them, instabilities in estimating many of the key parameters that are required for a correct 3D reconstruction that occur all along the processing workflow are referred to, which may significantly affect the reliability of the whole process. In the field, the term overfitting has been coined to refer to some particular kinds of artifacts. It is argued that overfitting is a statistical bias in key parameter-estimation steps in the 3D reconstruction process, including intrinsic algorithmic bias. It is also shown that common tools (Fourier shell correlation) and strategies (gold standard) that are normally used to detect or prevent overfitting do not fully protect against it. Alternatively, it is proposed that detecting the bias that leads to overfitting is much easier when addressed at the level of parameter estimation, rather than detecting it once the particle images have been combined into a 3D map. Comparing the results from multiple algorithms (or at least, independent executions of the same algorithm) can detect parameter bias. These multiple executions could then be averaged to give a lower variance estimate of the underlying parameters.
Assuntos
Imageamento Tridimensional , Viés , Consenso , Microscopia Crioeletrônica/métodos , Imageamento Tridimensional/métodos , Reprodutibilidade dos TestesRESUMO
Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed.
Assuntos
Processamento de Imagem Assistida por Computador , Imagem Individual de Molécula , Microscopia Crioeletrônica , Substâncias Macromoleculares , Fluxo de TrabalhoRESUMO
Electron tomography is a technique to obtain three-dimensional structural information of samples. However, the technique is limited by shifts occurring during acquisition that need to be corrected before the reconstruction process. In 2009, we proposed an approach for post-acquisition alignment of tilt series images. This approach was marker-free, based on patch tracking and integrated in free software. Here, we present improvements to the method to make it more reliable, stable and accurate. In addition, we modified the image formation model underlying the alignment procedure to include different deformations occurring during acquisition. We propose a new way to correct these computed deformations to obtain reconstructions with reduced artifacts. The new approach has demonstrated to improve the quality of the final 3D reconstruction, giving access to better defined structures for different transmission electron tomography methods: resin embedded STEM-tomography and cryo-TEM tomography. The method is freely available in TomoJ software.
RESUMO
In the last years, biological treatment plants for the previously separated organic fraction from municipal solid wastes (OFMSW) have gained importance. In these processes a liquid effluent (liquid fraction from the digestate and leachate from composting piles), which has to be treated previously to its discharge, is produced. In this paper, the characteristics of the mixed liquor from two full-scale membrane bioreactors treating the effluents of two OFMSW treatment plants have been evaluated in view to study their influence on membrane fouling in terms of filterability. For that, the mixed liquor samples have been ultrafiltrated in an UF laboratory plant. Besides, the effect of the influent characteristics to MBRs and the values of the chemical and physical parameters of the mixed liquors on the filterability have been studied. Results showed that the filterability of the mixed liquor was strongly influenced by the soluble microbial products in the mixed liquors and the influent characteristics to MBR. Permeate flux of MBR mixed liquor treating the most polluted wastewater was considerable the lowest (around 20 L/m(2) h for some samples), what was explained by viscosity and soluble microbial products concentration higher than those measured in other MBR mixed liquor.