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Understanding how structure and function meet to drive biological processes is progressively shifting the cryoEM field towards a more advanced analysis of macromolecular flexibility. Thanks to techniques such as single-particle analysis and electron tomography, it is possible to image a macromolecule in different states, information that can subsequently be extracted through advanced image-processing methods to build a richer approximation of a conformational landscape. However, the interoperability of all of these algorithms remains a challenging task that is left to users, preventing them from defining a single flexible workflow in which conformational information can be addressed by different algorithms. Therefore, in this work, a new framework integrated into Scipion is proposed called the Flexibility Hub. This framework automatically handles intercommunication between different heterogeneity software, simplifying the task of combining the software into workflows in which the quality and the amount of information extracted from flexibility analysis is maximized.

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Multi-color fluorescence imaging is a powerful tool for studying the spatial relationships and interactions among sub-cellular structures in biological specimens. However, if improperly corrected, geometrical distortions caused by mechanical drift, refractive index mismatch, or chromatic aberration can lead to lower image resolution. In this paper, we present an extension of the image processing framework of Scipion by integrating a protocol called OFM Corrector, which corrects geometrical distortions in real-time using a B-spline-based elastic continuous registration technique. Our proposal provides a simple strategy to overcome chromatic aberration by digitally re-aligning color channels in multi-color fluorescence microscopy images, even in 3D or time. Our method relies on a geometrical calibration, which we do with fluorescent beads excited by different wavelengths of light and subsequently registered to get the elastic warp as a reference to correct chromatic shift. Our software is freely available with a user-friendly GUI and can be broadly used for various biological imaging problems. The paper presents a valuable tool for researchers working in light microscopy facilities.

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Cryo electron microscopy (cryo-EM) instrumentation allows obtaining 3D reconstruction of the structure of biomolecular complexes in vitro (purified complexes studied by single particle analysis) and in situ (complexes studied in cells by cryo electron tomography). Standard cryo-EM approaches allow high-resolution reconstruction of only a few conformational states of a molecular complex, as they rely on data classification into a given number of classes to increase the resolution of the reconstruction from the most populated classes while discarding all other classes. Such discrete classification approaches result in a partial picture of the full conformational variability of the complex, due to continuous conformational transitions with many, uncountable intermediate states. In this article, we present the software with a user-friendly graphical interface for running two recently introduced methods, namely, MDSPACE and MDTOMO, to obtain continuous conformational landscapes of biomolecules by analyzing in vitro and in situ cryo-EM data (single particle images and subtomograms) based on molecular dynamics simulations of an available atomic model of one of the conformations. The MDSPACE and MDTOMO software is part of the open-source ContinuousFlex software package (starting from version 3.4.2 of ContinuousFlex), which can be run as a plugin of the Scipion software package (version 3.1 and later), broadly used in the cryo-EM field.

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ContinuousFlex is a user-friendly open-source software package for analyzing continuous conformational variability of macromolecules in cryo electron microscopy (cryo-EM) and cryo electron tomography (cryo-ET) data. In 2019, ContinuousFlex became available as a plugin for Scipion, an image processing software package extensively used in the cryo-EM field. Currently, ContinuousFlex contains software for running (1) recently published methods HEMNMA-3D, TomoFlow, and NMMD; (2) earlier published methods HEMNMA and StructMap; and (3) methods for simulating cryo-EM and cryo-ET data with conformational variability and methods for data preprocessing. It also includes external software for molecular dynamics simulation (GENESIS) and normal mode analysis (ElNemo), used in some of the mentioned methods. The HEMNMA software has been presented in the past, but not the software of other methods. Besides, ContinuousFlex currently also offers a deep learning extension of HEMNMA, named DeepHEMNMA. In this article, we review these methods in the context of the ContinuousFlex package, developed to facilitate their use by the community.

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Xmipp is an open-source software package consisting of multiple programs for processing data originating from electron microscopy and electron tomography, designed and managed by the Biocomputing Unit of the Spanish National Center for Biotechnology, although with contributions from many other developers over the world. During its 25 years of existence, Xmipp underwent multiple changes and updates. While there were many publications related to new programs and functionality added to Xmipp, there is no single publication on the Xmipp as a package since 2013. In this article, we give an overview of the changes and new work since 2013, describe technologies and techniques used during the development, and take a peek at the future of the package.

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Cryo-electron microscopy has established as a mature structural biology technique to elucidate the three-dimensional structure of biological macromolecules. The Coulomb potential of the sample is imaged by an electron beam, and fast semi-conductor detectors produce movies of the sample under study. These movies have to be further processed by a whole pipeline of image-processing algorithms that produce the final structure of the macromolecule. In this chapter, we illustrate this whole processing pipeline putting in value the strength of "meta algorithms," which are the combination of several algorithms, each one with different mathematical rationale, in order to distinguish correctly from incorrectly estimated parameters. We show how this strategy leads to superior performance of the whole pipeline as well as more confident assessments about the reconstructed structures. The "meta algorithms" strategy is common to many fields and, in particular, it has provided excellent results in bioinformatics. We illustrate this combination using the workflow engine, Scipion.

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Scipion is a modular image-processing framework that integrates several software packages under a unified interface while taking care of file formats and conversions. Here, new developments and capabilities of the Scipion plugin for the widely used RELION software package are presented and illustrated with an image-processing pipeline for published data. The user interfaces of Scipion and RELION are compared and the key differences are highlighted, allowing this manuscript to be used as a guide for both new and experienced users of this software. Different on-the-fly image-processing options are also discussed, demonstrating the flexibility of the Scipion framework.

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Electron microscopy of macromolecular structures is an approach that is in increasing demand in the field of structural biology. The automation of image acquisition has greatly increased the potential throughput of electron microscopy. Here, the focus is on the possibilities in Scipion to implement flexible and robust image-processing workflows that allow the electron-microscope operator and the user to monitor the quality of image acquisition, assessing very simple acquisition measures or obtaining a first estimate of the initial volume, or the data resolution and heterogeneity, without any need for programming skills. These workflows can implement intelligent automatic decisions and they can warn the user of possible acquisition failures. These concepts are illustrated by analysis of the well known 2.2 Šresolution ß-galactosidase data set.

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Three dimensional electron microscopy is becoming a very data-intensive field in which vast amounts of experimental images are acquired at high speed. To manage such large-scale projects, we had previously developed a modular workflow system called Scipion (de la Rosa-Trevín et al., 2016). We present here a major extension of Scipion that allows processing of EM images while the data is being acquired. This approach helps to detect problems at early stages, saves computing time and provides users with a detailed evaluation of the data quality before the acquisition is finished. At present, Scipion has been deployed and is in production mode in seven Cryo-EM facilities throughout the world.

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