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

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

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

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

Dendritic cell deficiencies persist seven months after SARS-CoV-2 infection

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 infection induces an exacerbated inflammation driven by innate immunity components. Dendritic cells (DCs) play a key role in the defense against viral infections, for instance plasmacytoid DCs (pDCs), have the capacity to produce vast amounts of interferon-alpha (IFN-α). In COVID-19 there is a deficit in DC numbers and IFN-α production, which has been associated with disease severity. In this work, we described that in addition to the DC deficiency, several DC activation and homing markers were altered in acute COVID-19 patients, which were associated with multiple inflammatory markers. Remarkably, previously hospitalized and nonhospitalized patients remained with decreased numbers of CD1c+ myeloid DCs and pDCs seven months after SARS-CoV-2 infection. Moreover, the expression of DC markers such as CD86 and CD4 were only restored in previously nonhospitalized patients, while no restoration of integrin β7 and indoleamine 2,3-dyoxigenase (IDO) levels were observed. These findings contribute to a better understanding of the immunological sequelae of COVID-19

SARS-CoV-2 viral load in nasopharyngeal swabs is not an independent predictor of unfavorable outcome

The aim was to assess the ability of nasopharyngeal SARS-CoV-2 viral load at first patient’s hospital evaluation to predict unfavorable outcomes. We conducted a prospective cohort study including 321 adult patients with confirmed COVID-19 through RT-PCR in nasopharyngeal swabs. Quantitative Synthetic SARS-CoV-2 RNA cycle threshold values were used to calculate the viral load in log10 copies/mL. Disease severity at the end of follow up was categorized into mild, moderate, and severe. Primary endpoint was a composite of intensive care unit (ICU) admission and/or death (n = 85, 26.4%). Univariable and multivariable logistic regression analyses were performed. Nasopharyngeal SARS-CoV-2 viral load over the second quartile (≥ 7.35 log10 copies/mL, p = 0.003) and second tertile (≥ 8.27 log10 copies/mL, p = 0.01) were associated to unfavorable outcome in the unadjusted logistic regression analysis. However, in the final multivariable analysis, viral load was not independently associated with an unfavorable outcome. Five predictors were independently associated with increased odds of ICU admission and/or death: age ≥ 70 years, SpO2, neutrophils > 7.5 × 103/µL, lactate dehydrogenase ≥ 300 U/L, and C-reactive protein ≥ 100 mg/L. In summary, nasopharyngeal SARS-CoV-2 viral load on admission is generally high in patients with COVID-19, regardless of illness severity, but it cannot be used as an independent predictor of unfavorable clinical outcome

Embryonic and naupliar development of Eudiaptomus gracilis and Eudiaptomus graciloides at different temperatures: comments on individual variability

Eudiaptomus gracilis and Eudiaptomus graciloides are amongst the most common calanoid copepods in Europe and co-occur in many lakes. To understand their ecological dynamics, it is essential to know their responses to environmental variation. The finding that E. graciloides exhibits diapause in winter, whereas E. gracilis reproduces throughout the year indicates that the two species might differ in their temperature responses. We measured embryonic and naupliar development times, clutch size (number of eggs per sac), hatching percentage and body length of the first copepodid stage (CI) of both species under non-limiting food conditions at different temperatures. Special attention was given to individual variability on development times and to the use of the gamma density function (GDF) for fitting the probability of moulting. Results show that E. gracilis exhibits just slightly faster development times, lower individual variability, higher hatching percentage and larger clutches than E. graciloides and that extreme temperatures affected E. gracilis less intensely than E. graciloides. GDF was a good tool for estimating individual variability in the different experiments

Agricultural impacts on Mediterranean wetlands : the effect of pesticides on survival and hatching rates in copepods

Wetlands are one of the most altered natural systems due to the creation and development of agricultural landscapes. Some of agriculture’s impacts are in relation to water quality decreases, due to the use of potentially toxic herbicides or pesticides, and they are responsible of ecological alterations. This study shows the negative effect that two pesticides generate in a population of the copepod Arctodiaptomus salinus in an aquatic ecosystem that is surrounded by intensive olive tree cultivation. Adult females and egg sacs of that calanoid copepod were exposed to different concentrations of copper sulphate and the pesticide dimethoate, to examine their tolerance response. The adult lethal concentration obtained was lower than the regular dose of pesticide used in olive agriculture. These results also reflect the negative effect over A. salinus secondary production as a consequence of the increase in females and nauplii mortality and by the hatching rate reduction