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

Efecto de un tratamiento previo por altas presiones hidrostáticas sobre la alteración lipídica de jurel chileno (Trachurus murphyi) refrigerado

Lipid damage evolution was analyzed in chilled Chilean jack mackerel (Trachurus murphyi) previously treated with high hydrostatic pressure (HHP) technology. Different pressure levels and pressure holding times were tested. In addition, fish corresponding to pre- and post-rigor mortis (RM) stages were comparatively studied. Previous HHP treatment led to a marked lipid hydrolysis inhibition in chilled fish. Increasing the pressure level and pressure holding time led to a lower free fatty acid content, with the effect of pressure being more relevant. According to the analysis of different types of lipid oxidation indexes, no effect of the previous HHP treatment on the lipid oxidation development could be determined in chilled jack mackerel. Concerning the effect of the RM stage of raw fish, a higher primary and secondary lipid oxidation development was observed in fish corresponding to the post-RM condition throughout the chilled storage; although a definite effect on lipid hydrolysis could not be found.Se estudió la evolución de la alteración lipídica en jurel chileno (Trachurus murphyi) refrigerado previamente tratado a altas presiones hidrostáticas (HHP). Se aplicaron distintos valores de presión y tiempo de presurización; asimismo, se analizó de forma comparativa la respuesta al proceso del pescado inicial en estados pre- y post-rigor mortis (RM). El tratamiento previo por HHP produjo inhibición de la hidrólisis lipídica en pescado refrigerado, siendo más intenso el efecto de la presión que el del tiempo de presurización. De acuerdo con el análisis de distintos índices de oxidación, no se concluyó un efecto determinante sobre la oxidación lipídica por parte del tratamiento previo de HHP. En relación al efecto del estado de RM del pescado inicial, se observó una oxidación primaria y secundaria mayor en jurel correspondiente a la condición post-RM durante la conservación en refrigeración; sin embargo, no se detectó un efecto claro sobre la hidrólisis lipídica

Flexible workflows for on-the-fly electronmicroscopy single-particle image processing using Scipion

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 Å resolution β-galactosidase data setThe authors would like to acknowledge financial support from The Spanish Ministry of Economy and Competitiveness through the BIO2016-76400-R (AEI/FEDER, UE) grant, the Comunidad Auto´noma de Madrid through grant S2017/BMD3817, the Instituto de Salud Carlos III (PT17/0009/0010), the European Union (EU) and Horizon 2020 through the CORBEL grant (INFRADEV-1-2014-1, Proposal 654248), the ‘la Caixa’ Foundation (ID 100010434, Fellow LCF/BQ/ IN18/11660021), Elixir–EXCELERATE (INFRADEV-3- 2015, Proposal 676559), iNEXT (INFRAIA-1-2014-2015, Proposal 653706), EOSCpilot (INFRADEV-04-2016, Proposal 739563) and INSTRUCT–ULTRA (INFRADEV03-2016-2017, Proposal 731005

Cryo-EM and single-particle analysis with Scipion

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

Algorithmic robustness to preferred orientations in single particle analysis by CryoEM

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 achievedWe acknowledge support from “la Caixa” Foundation (Fellowship LCF/BQ/DI18/11660021. This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 713673. We also thank the financial support from the Spanish Ministry of Economy and Competitiveness through Grants BIO2016-76400-R(AEI/FEDER, UE) and SEV 2017-0712, the “Comunidad Autónoma de Madrid” through Grant: S2017/BMD-3817, Instituto de Salud Carlos III, PT17/ 0009/0010 (ISCIII-SGEFI/ERDF), European Union (EU) and Horizon 2020 through grants: CORBEL (INFRADEV-1-2014-1, Proposal: 654248), INSTRUCT-ULTRA (INFRADEV-03-2016-2017, Proposal: 731005), EOSC Life (INFRAEOSC-04-2018, Proposal: 824087), High- ResCells (ERC-2018-SyG, Proposal: 810057), IMpaCT (WIDESPREAD-03-2018 – Proposal: 857203), EOSC-Synergy (EINFRA-EOSC-5, Proposal: 857647), and iNEXT-Discovery (Proposal: 871037). The authors acknowledge the support and the use of resources of Instruct, a Landmark ESFRI projec

Survey of the analysis of continuous conformational variability of biological macromolecules by electron microscopy

Single-particle analysis by electron microscopy is a well established technique for analyzing the three-dimensional structures of biological macromolecules. Besides its ability to produce high-resolution structures, it also provides insights into the dynamic behavior of the structures by elucidating their conformational variability. Here, the different image-processing methods currently available to study continuous conformational changes are reviewedThe authors would like to acknowledge support from the Spanish Ministry of Economy and Competitiveness through grants BIO2013-44647-R and BIO2016-76400-R (AEI/ FEDER, UE), Comunidad Autonoma de Madrid through grant S2017/BMD-3817, Instituto de Salud Carlos III through grants PT13 /0001/0009 and PT17/0009/0010,the European Union (EU) and Horizon 2020 through West-Life (EINFRA- 2015-1, Proposal 675858), CORBEL (INFRADEV-1-2014-1, Proposal 654248), ELIXIR–EXCELERATE (INFRADEV-3- 2015, Proposal 676559), iNEXT (INFRAIA-1-2014-2015, Proposal 653706), EOSCpilot (INFRADEV-04-2016, Proposal 739563) and the National Institutes of Health (P41 GM 103712) (IB

Th17-Related Genes and Celiac Disease Susceptibility

Th17 cells are known to be involved in several autoimmune or inflammatory diseases. In celiac disease (CD), recent studies suggest an implication of those cells in disease pathogenesis. We aimed at studying the role of genes relevant for the Th17 immune response in CD susceptibility. A total of 101 single nucleotide polymorphisms (SNPs), mainly selected to cover most of the variability present in 16 Th17-related genes (IL23R, RORC, IL6R, IL17A, IL17F, CCR6, IL6, JAK2, TNFSF15, IL23A, IL22, STAT3, TBX21, SOCS3, IL12RB1 and IL17RA), were genotyped in 735 CD patients and 549 ethnically matched healthy controls. Case-control comparisons for each SNP and for the haplotypes resulting from the SNPs studied in each gene were performed using chi-square tests. Gene-gene interactions were also evaluated following different methodological approaches. No significant results emerged after performing the appropriate statistical corrections. Our results seem to discard a relevant role of Th17 cells on CD risk

Antimicrobial therapy for acute cholangitis: Tokyo Guidelines

Antimicrobial agents should be administered to all patients with suspected acute cholangitis as a priority as soon as possible. Bile cultures should be performed at the earliest opportunity. The important factors which should be considered in selecting antimicrobial therapy include the agent’s activity against potentially infecting bacteria, the severity of the cholangitis, the presence or absence of renal and hepatic diseases, the patient’s recent history of antimicrobial therapy, and any recent culture results, if available. Biliary penetration of the microbial agents should also be considered in the selection of antimicrobials, but activity against the infecting isolates is of greatest importance. If the causative organisms are identified, empirically chosen antimicrobial drugs should be replaced by narrower-spectrum antimicrobial agents, the most appropriate for the species and the site of the infection