Scipion: a software framework toward integration, reproducibility and validation in 3D Electron Microscopy

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Escuela Politécnica Superior, Departamento de Ingeniería Informática. Fecha de lectura : 25-10-2017In the past few years, 3D electron microscopy (3DEM) has undergone a revolution in instrumentation and methodology. One of the central players in this wide-reaching change is the continuous development of image processing software. Here we present Scipion, a software framework for integrating several 3DEM software packages through a work owbased approach. Scipion allows the execution of reusable, standardized, traceable and reproducible image-processing protocols. These protocols incorporate tools from di erent programs while providing full interoperability among them. Scipion is an open-source project that can be downloaded from http://scipion.cnb.csic.es

Cryo-EM and the elucidation of new macromolecular structures: Random Conical Tilt revisited

Cryo-Electron Microscopy (cryo-EM) of macromolecular complexes is a fundamental structural biology technique which is expanding at a very fast pace. Key to its success in elucidating the three-dimensional structure of a macromolecular complex, especially of small and non-symmetric ones, is the ability to start from a low resolution map, which is subsequently refined with the actual images collected at the microscope. There are several methods to produce this first structure. Among them, Random Conical Tilt (RCT) plays a prominent role due to its unbiased nature (it can create an initial model based on experimental measurements). In this article, we revise the fundamental mathematical expressions supporting RCT, providing new expressions handling all key geometrical parameters without the need of intermediate operations, leading to improved automation and overall reliability, essential for the success of cryo-EM when analyzing new complexes. We show that the here proposed RCT workflow based on the new formulation performs very well in practical cases, requiring very few image pairs (as low as 13 image pairs in one of our examples) to obtain relevant 3D maps.We thank Dr. Llorca for his support during the acquisition of the C3b images and Dr. Shaikh for his support in the use of Spider for the RCT reconstructions. The authors would like to acknowledge economical support from the Spanish Ministry of Economy and Competitiveness through grants AIC-A-2011-0638 and BIO2013-44647-R, the Comunidad de Madrid through grant CAM (S2010/BMD-2305), as well as a postdoctoral Juan de la Cierva grant with reference JCI-2011-10185 to Javier Vargas. Vahid Abrishami is a holder of La Caixa scholarship and C.O.S. Sorzano is recipient of a Ramon y Cajal fellowship

Hybrid Electron Microscopy Normal Mode Analysis graphical interface and protocol

International audienceThis article presents an integral graphical interface to the Hybrid Electron Microscopy Normal Mode Analysis (HEMNMA) approach that was developed for capturing continuous motions of large macromolecular complexes from single-particle EM images. HEMNMA was shown to be a good approach to analyze multiple conformations of a macromolecular complex but it could not be widely used in the EM field due to a lack of an integral interface. In particular, its use required switching among different software sources as well as selecting modes for image analysis was difficult without the graphical interface. The graphical interface was thus developed to simplify the practical use of HEMNMA. It is implemented in the open-source software package Xmipp 3.1 (http://xmipp.cnb.csic.es) and only a small part of it relies on MATLAB that is accessible through the main interface. Such integration provides the user with an easy way to perform the analysis of macromolecular dynamics and forms a direct connection to the single-particle reconstruction process. A step-by-step HEMNMA protocol with the graphical interface is given in full details in Supplementary material. The graphical interface will be useful to experimentalists who are interested in studies of continuous conformational changes of macromolecular complexes beyond the modeling of continuous heterogeneity in single particle reconstruction

Localized reconstruction in Scipion expedites the analysis of symmetry mismatches in cryo-EM data

Technological advances in transmission electron microscopes and detectors have turned cryogenic electron microscopy (cryo-EM) into an essential tool for structural biology. A commonly used cryo-EM data analysis method, single particle analysis, averages hundreds of thousands of low-dose images of individual macromolecular complexes to determine a density map of the complex. The presence of symmetry in the complex is beneficial since each projection image can be assigned to multiple views of the complex. However, data processing that applies symmetry can average out asymmetric features and consequently data analysis methods are required to resolve asymmetric structural features. Scipion is a cryo-EM image processing framework that integrates functionalities from different image processing packages as plugins. To extend its functionality for handling symmetry mismatches, we present here a Scipion plugin termed LocalRec implementing the localized reconstruction method. When tested on an adenovirus data set, the plugin enables resolving the symmetry-mismatched trimeric fibre bound to the five-fold vertices of the capsid. Furthermore, it improves the structure determination of the icosahedral capsid by dealing with the defocus gradient across the particle. LocalRec is expected to be widely applicable in a range of cryo-EM investigations of flexible and symmetry mismatched complexes.Peer reviewe

Iterative elastic 3D-to-2D alignment method using normal modes for studying structural dynamics of large macromolecular complexes

This article presents a method to study large-scale conformational changes by combining electron microscopy (EM) single-particle image analysis and normal mode analysis (NMA). It is referred to as HEMNMA, which stands for hybrid electron microscopy normal mode analysis. NMA of a reference structure (atomic-resolution structure or EM volume) is used to predict possible motions that are then confronted with EM images within an automatic iterative elastic 3D-to-2D alignment procedure to identify actual motions in the imaged samples. HEMNMA can be used to extensively analyze the conformational changes and may be used in combination with classic discrete procedures. The identified conformations allow modeling of deformation pathways compatible with the experimental data. HEMNMA was tested with synthetic and experimental data sets of E. coli 70S ribosome, DNA polymerase Pol α and B subunit complex of the eukaryotic primosome, and tomato bushy stunt virus. © 2014 Elsevier Ltd.This work was supported by the French National Center for Scientific Research and the Spanish National Research Council (CSIC2009FR0015 and PICS 2011 to S.J. and C.O.S.S.); the Spanish Ministry of Economy and Competitiveness (ACI2009-10220, ACI2010-1088, and BIO2010-16566 to J.M. Carazo and SAF2011-22988 to O.L.); the European Social Fund and the Ministerio de Educación y Ciencia (“Ramón y Cajal” fellowship to C.O.S.S. and J.R.B.-C.).Peer Reviewe

Iterative Elastic 3D-to-2D Alignment Method Using Normal Modes for Studying Structural Dynamics of Large Macromolecular Complexes

International audienceThis article presents a method to study large-scale conformational changes by combining electron microscopy (EM) single-particle image analysis and normal mode analysis (NMA). It is referred to as HEMNMA, which stands for hybrid electron microscopy normal mode analysis. NMA of a reference structure (atomic-resolution structure or EM volume) is used to predict possible motions that are then confronted with EM images within an automatic iterative elastic 3D-to-2D alignment procedure to identify actual motions in the imaged samples. HEMNMA can be used to extensively analyze the conformational changes and may be used in combination with classic discrete procedures. The identified conformations allow modeling of deformation pathways compatible with the experimental data. HEMNMA was tested with synthetic and experimental data sets of E. coli 70S ribosome, DNA polymerase Pol a and B subunit complex of the eukaryotic primosome, and tomato bushy stunt virus

ScipionCloud: An integrative and interactive gateway for large scale cryo electron microscopy image processing on commercial and academic clouds

New instrumentation for cryo electron microscopy (cryoEM) has significantly increased data collection rate as well as data quality, creating bottlenecks at the image processing level. Current image processing model of moving the acquired images from the data source (electron microscope) to desktops or local clusters for processing is encountering many practical limitations. However, computing may also take place in distributed and decentralized environments. In this way, cloud is a new form of accessing computing and storage resources on demand. Here, we evaluate on how this new computational paradigm can be effectively used by extending our current integrative framework for image processing, creating ScipionCloud. This new development has resulted in a full installation of Scipion both in public and private clouds, accessible as public “images”, with all the required preinstalled cryoEM software, just requiring a Web browser to access all Graphical User Interfaces. We have profiled the performance of different configurations on Amazon Web Services and the European Federated Cloud, always on architectures incorporating GPU’s, and compared them with a local facility. We have also analyzed the economical convenience of different scenarios, so cryoEM scientists have a clearer picture of the setup that is best suited for their needs and budgets.Spanish Ministry of Economy and Competitiveness through Grants AIC-A-2011-0638, BIO2016-76400-R and BFU2016-74868-P. This work was co-funded by the European Union (EU) and Horizon 2020 through grant West-Life (EINFRA-2015-1, Proposal: 675858) and the EGI-Engage project under Grant number 654142.Peer reviewe