MDSPACE: Extracting Continuous Conformational Landscapes from Cryo-EM Single Particle Datasets Using 3D-to-2D Flexible Fitting based on Molecular Dynamics Simulation.

This article presents an original approach for extracting atomic-resolution landscapes of continuous conformational variability of biomolecular complexes from cryo electron microscopy (cryo-EM) single particle images. This approach is based on a new 3D-to-2D flexible fitting method, which uses molecular dynamics (MD) simulation and is embedded in an iterative conformational-landscape refinement scheme. This new approach is referred to as MDSPACE, which stands for Molecular Dynamics simulation for Single Particle Analysis of Continuous Conformational hEterogeneity. The article describes the MDSPACE approach and shows its performance using synthetic and experimental datasets.

MDSPACE and MDTOMO Software for Extracting Continuous Conformational Landscapes from Datasets of Single Particle Images and Subtomograms Based on Molecular Dynamics Simulations: Latest Developments in ContinuousFlex Software Package.

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.

ContinuousFlex: Software package for analyzing continuous conformational variability of macromolecules in cryo electron microscopy and tomography data.

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.

TomoFlow: Analysis of Continuous Conformational Variability of Macromolecules in Cryogenic Subtomograms based on 3D Dense Optical Flow.

Cryogenic Electron Tomography (cryo-ET) allows structural and dynamics studies of macromolecules in situ. Averaging different copies of imaged macromolecules is commonly used to obtain their structure at higher resolution and discrete classification to analyze their dynamics. Instrumental and data processing developments are progressively equipping cryo-ET studies with the ability to escape the trap of classification into a complete continuous conformational variability analysis. In this work, we propose TomoFlow, a method for analyzing macromolecular continuous conformational variability in cryo-ET subtomograms based on a three-dimensional dense optical flow (OF) approach. The resultant lower-dimensional conformational space allows generating movies of macromolecular motion and obtaining subtomogram averages by grouping conformationally similar subtomograms. The animations and the subtomogram group averages reveal accurate trajectories of macromolecular motion based on a novel mathematical model that makes use of OF properties. This paper describes TomoFlow with tests on simulated datasets generated using different techniques, namely Normal Mode Analysis and Molecular Dynamics Simulation. It also shows an application of TomoFlow on a dataset of nucleosomes in situ, which provided promising results coherent with previous findings using the same dataset but without imposing any prior knowledge on the analysis of the conformational variability. The method is discussed with its potential uses and limitations.

Advances in Xmipp for Cryo-Electron Microscopy: From Xmipp to Scipion.

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.

HEMNMA-3D: Cryo Electron Tomography Method Based on Normal Mode Analysis to Study Continuous Conformational Variability of Macromolecular Complexes.

Cryogenic electron tomography (cryo-ET) allows structural determination of biomolecules in their native environment (in situ). Its potential of providing information on the dynamics of macromolecular complexes in cells is still largely unexploited, due to the challenges of the data analysis. The crowded cell environment and continuous conformational changes of complexes make difficult disentangling the data heterogeneity. We present HEMNMA-3D, which is, to the best of our knowledge, the first method for analyzing cryo electron subtomograms in terms of continuous conformational changes of complexes. HEMNMA-3D uses a combination of elastic and rigid-body 3D-to-3D iterative alignments of a flexible 3D reference (atomic structure or electron microscopy density map) to match the conformation, orientation, and position of the complex in each subtomogram. The elastic matching combines molecular mechanics simulation (Normal Mode Analysis of the 3D reference) and experimental, subtomogram data analysis. The rigid-body alignment includes compensation for the missing wedge, due to the limited tilt angle of cryo-ET. The conformational parameters (amplitudes of normal modes) of the complexes in subtomograms obtained through the alignment are processed to visualize the distribution of conformations in a space of lower dimension (typically, 2D or 3D) referred to as space of conformations. This allows a visually interpretable insight into the dynamics of the complexes, by calculating 3D averages of subtomograms with similar conformations from selected (densest) regions and by recording movies of the 3D reference's displacement along selected trajectories through the densest regions. We describe HEMNMA-3D and show its validation using synthetic datasets. We apply HEMNMA-3D to an experimental dataset describing in situ nucleosome conformational variability. HEMNMA-3D software is available freely (open-source) as part of ContinuousFlex plugin of Scipion V3.0 (http://scipion.i2pc.es).

Methamphetamine drug abuse and addiction: Effects on face asymmetry.

The abnormal aging mechanism associated with drug abuse results in poor performance of face recognition systems on illicit drug addicts (mainly methamphetamine). Consequently, the high correlation between drug addiction and crime exaggerates the urge for further investigations to originate and overcome this problem. Concurrently, face asymmetry was found to play a significant role in face recognition and age estimation. Therefore, facial asymmetry assessment for meth-addicts is highly serviceable, acknowledging how meth addiction accelerates biological aging and causes severe face distortion. In this work, we address facial asymmetry for meth-addicts compared with ordinary people. We assess facial asymmetry by employing the most credible state-of-the-art tools for local and global two-dimensional (2D) methods. More specifically, we use a classical bilateral-based metric for local analysis, combined with a proposed global approach, that we refer to as the Area Mismatch metric, to give a vivid overview of geometrical facial asymmetry. Finally, we construct a metric for textural facial asymmetry assessment by employing the Structural Similarity Index (SSIM) for dual regions in a given face. We apply the aforementioned metrics on two databases, a recently collected meth-addicted database and a regular aging database (FERET). Statistical analysis indicated a significant increment of facial asymmetry for meth addicts while aging, three to five times more than ordinary people. This study definitively answers the question regarding the correlation between meth abuse and addiction and the increase of facial asymmetry. Also, it confirms previous findings concerning aging and increased facial asymmetry.

Hybrid Electron Microscopy Normal Mode Analysis with Scipion.

Hybrid Electron Microscopy Normal Mode Analysis (HEMNMA) method was introduced in 2014. HEMNMA computes normal modes of a reference model (an atomic structure or an electron microscopy map) of a molecular complex and uses this model and its normal modes to analyze single-particle images of the complex to obtain information on its continuous conformational changes, by determining the full distribution of conformational variability from the images. An advantage of HEMNMA is a simultaneous determination of all parameters of each image (particle conformation, orientation, and shift) through their iterative optimization, which allows applications of HEMNMA even when the effects of conformational changes dominate those of orientational changes. HEMNMA was first implemented in Xmipp and was using MATLAB for statistical analysis of obtained conformational distributions and for fitting of underlying trajectories of conformational changes. A HEMNMA implementation independent of MATLAB is now available as part of a plugin of Scipion V2.0 (http://scipion.i2pc.es). This plugin, named ContinuousFlex, can be installed by following the instructions at https://pypi.org/project/scipion-em-continuousflex. In this article, we present this new HEMNMA software, which is user-friendly, totally free, and open-source. STATEMENT FOR A BROADER AUDIENCE: This article presents Hybrid Electron Microscopy Normal Mode Analysis (HEMNMA) software that allows analyzing single-particle images of a complex to obtain information on continuous conformational changes of the complex, by determining the full distribution of conformational variability from the images. The HEMNMA software is user-friendly, totally free, open-source, and available as part of ContinuousFlex plugin (https://pypi.org/project/scipion-em-continuousflex) of Scipion V2.0 (http://scipion.i2pc.es).