88 research outputs found
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
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 2.2â
Ă
resolution ÎČ-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 AutoÂŽnoma 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 âla Caixaâ Foundation (ID 100010434, Fellow LCF/BQ/
IN18/11660021), ElixirâEXCELERATE (INFRADEV-3-
2015, Proposal 676559), iNEXT (INFRAIA-1-2014-2015,
Proposal 653706), EOSCpilot (INFRADEV-04-2016,
Proposal 739563) and INSTRUCTâULTRA (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
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), ELIXIRâEXCELERATE (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 âla Caixaâ Foundation (Fellowship LCF/BQ/DI18/11660021. This project has received funding from the European Unionâs 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 âComunidad AutĂłnoma de Madridâ 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 â 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
Near-atomic cryo-EM structure of PRC1 bound to the microtubule
Proteins that associate with microtubules (MTs) are crucial to generate MT arrays and establish different cellular architectures. One example is PRC1 (protein regulator of cytokinesis 1), which cross-links antiparallel MTs and is essential for the completion of mitosis and cytokinesis. Here we describe a 4-Ă
-resolution cryo-EM structure of monomeric PRC1 bound to MTs. Residues in the spectrin domain of PRC1 contacting the MT are highly conserved and interact with the same pocket recognized by kinesin. We additionally found that PRC1 promotes MT assembly even in the presence of the MT stabilizer taxol. Interestingly, the angle of the spectrin domain on the MT surface corresponds to the previously observed cross-bridge angle between MTs cross-linked by full-length, dimeric PRC1. This finding, together with molecular dynamic simulations describing the intrinsic flexibility of PRC1, suggests that the MT-spectrin domain interface determines the geometry of the MT arrays cross-linked by PRC1.Peer Reviewe
Re-examining the spectra of macromolecules. Current practice of spectral quasi B-factor flattening.
The analysis of structure factors in 3D cryo-EM Coulomb potential maps and their âenhancementâ at the end of the reconstruction process is a well-established practice, normally referred to as sharpening. The aim is to increase contrast and, in this way, to help tracing the atomic model. The most common way to accomplish this enhancement is by means of the so-called B-factor correction, which applies a global filter to boost high frequencies with some dampening considerations related to noise amplification. The results are maps with a better visual aspect and a quasiflat spectrum at medium and high frequencies. This practice is so widespread that most map depositions in the Electron Microscopy Data Base (EMDB) only contain sharpened maps. Here, the use in cryoEM of global B-factor corrections is theoretically and experimentally analyzed. Results clearly illustrate that protein spectra present a falloff. Thus, spectral quasi-flattening may produce protein spectra with distortions when compared with experimental ones, this fact, combined with the practice of reporting only sharpened maps, generates a sub-optimal situation in terms of data preservation, reuse and reproducibility. Now that the field is more advanced, we put forward two suggestions: (1) to use methods which keep more faithfully the original experimental signal properties of macromolecules when âenhancingâ the map, and (2) to further stress the need to deposit the original experimental maps without any postprocessing or sharpening, not only the enhanced maps. In the absence of access to these original maps data is lost, preventing their future analysis with new methods.The authors would like to acknowledge economical support from The Spanish Ministry of Economy and Competitiveness through Grants BIO2016-76400-R(AEI/FEDER, UE), the âComunidad AutĂłnoma de Madridâ through Grant: S2017/BMD-3817
Using Scipion for stream image processing at Cryo-EM facilities
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-TrevĂn 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.Spanish Ministry of Economy and Competitiveness through Grants BIO2016-76400R(AEI/FEDER, UE) and AEI/FEDER BFU 2016 74868P, the Comunidad AutĂłnoma de Madrid through Grant: S2017/BMD-3817, European Union (EU) and Horizon 2020 through grant Corbel (INFRADEV-1-2014-1, Proposal: 654248). The âKnut & Alice Wallenberg Foundationâ, and âA Pilot Facility development grant from Science for Life Laboratoryâ. European Union (EU) and Horizon 2020 through grant EOSCpilot (INFRADEV-04-2016, Proposal: 739563).This work used the EGI Infrastructure and is co-funded by the EGIEngage project (Horizon 2020) under Grant No. 654142. European Union (EU) and Horizon 2020 through grant West-Life (EINFRA-2015-1, Proposal: 675858) European Union (EU) and Horizon 2020 through grant Elixir - EXCELERATE (INFRADEV-3-2015, Proposal: 676559) European Union (EU) and Horizon 2020 through grant iNEXT (INFRAIA-1-2014-2015, Proposal: 653706).Peer reviewe
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