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

Particle alignment reliability in single particle electron cryomicroscopy: A general approach

Electron Microscopy is reaching new capabilities thanks to the combined effect of new technologies and new image processing methods. However, the reconstruction process is still complex, requiring many steps and elaborated optimization procedures. Therefore, the possibility to reach a wrong structure exists, justifying the need of robust statistical tests. In this work, we present a conceptually simple alignment test, which does not require tilt-pair images, to evaluate the alignment consistency between a set of projection images with respect to a given 3D density map. We test the approach on a number of problems in 3DEM, especially the ranking and evaluation of initial 3D volumes and high resolution 3D maps, where we show its usefulness in providing an objective evaluation for maps that have recently been subject to a strong controversy in the field. Additionally, this alignment statistical test can be linked to the early stages of structure solving of new complexes, streamlining the whole process.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), and from "Comfuturo" grant funded by the Fundacion General CSIC. C.O.S. Sorzano is recipient of a Ramon y Cajal fellowship. We acknowledge discussions with Sriram Subramanian on validation approaches related to the HIV trimer structure

Contourlet textual features: Improving the diagnosis of solitary pulmonary nodules in two dimensional ct images

Materials and Methods: A total of 6,299 CT images were acquired from 336 patients, with 1,454 benign pulmonary nodule images from 84 patients (50 male, 34 female) and 4,845 malignant from 252 patients (150 male, 102 female). Further to this, nineteen patient information categories, which included seven demographic parameters and twelve morphological features, were also collected. A contourlet was used to extract fourteen types of textural features. These were then used to establish three support vector machine models. One comprised a database constructed of nineteen collected patient information categories, another included contourlet textural features and the third one contained both sets of information. Ten-fold cross-validation was used to evaluate the diagnosis results for the three databases, with sensitivity, specificity, accuracy, the area under the curve (AUC), precision, Youden index, and F-measure were used as the assessment criteria. In addition, the synthetic minority over-sampling technique (SMOTE) was used to preprocess the unbalanced data.Results: Using a database containing textural features and patient information, sensitivity, specificity, accuracy, AUC, precision, Youden index, and F-measure were: 0.95, 0.71, 0.89, 0.89, 0.92, 0.66, and 0.93 respectively. These results were higher than results derived using the database without textural features (0.82, 0.47, 0.74, 0.67, 0.84, 0.29, and 0.83 respectively) as well as the database comprising only textural features (0.81, 0.64, 0.67, 0.72, 0.88, 0.44, and 0.85 respectively). Using the SMOTE as a pre-processing procedure, new balanced database generated, including observations of 5,816 benign ROIs and 5,815 malignant ROIs, and accuracy was 0.93.Objective: To determine the value of contourlet textural features obtained from solitary pulmonary nodules in two dimensional CT images used in diagnoses of lung cancer. Copyright:Conclusion: Our results indicate that the combined contourlet textural features of solitary pulmonary nodules in CT images with patient profile information could potentially improve the diagnosis of lung cancer

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

Electron Microscopy Structural Insights into CPAP Oligomeric Behavior:A Plausible Assembly Process of a Supramolecular Scaffold of the Centrosome

Centrosomal P4.1-associated protein (CPAP) is a cell cycle regulated protein fundamental for centrosome assembly and centriole elongation. In humans, the region between residues 897–1338 of CPAP mediates interactions with other proteins and includes a homodimerization domain. CPAP mutations cause primary autosomal recessive microcephaly and Seckel syndrome. Despite of the biological/clinical relevance of CPAP, its mechanistic behavior remains unclear and its C-terminus (the G-box/TCP domain) is the only part whose structure has been solved. This situation is perhaps due in part to the challenges that represent obtaining the protein in a soluble, homogeneous state for structural studies. Our work constitutes a systematic structural analysis on multiple oligomers of HsCPAP897−1338, using single-particle electron microscopy (EM) of negatively stained (NS) samples. Based on image classification into clearly different regular 3D maps (putatively corresponding to dimers and tetramers) and direct observation of individual images representing other complexes of HsCPAP897−1338 (i.e., putative flexible monomers and higher-order multimers), we report a dynamic oligomeric behavior of this protein, where different homo-oligomers coexist in variable proportions. We propose that dimerization of the putative homodimer forms a putative tetramer which could be the structural unit for the scaffold that either tethers the pericentriolar material to centrioles or promotes procentriole elongation. A coarse fitting of atomic models into the NS 3D maps at resolutions around 20 Å is performed only to complement our experimental data, allowing us to hypothesize on the oligomeric composition of the different complexes. In this way, the current EM work represents an initial step toward the structural characterization of different oligomers of CPAP, suggesting further insights to understand how this protein works, contributing to the elucidation of control mechanisms for centriole biogenesis.This work was supported by the Comunidad de Madrid through grant CAM (S2010/BMD- 2305) and the Spanish Ministry of Economy and Competitiveness through Grants AIC-A-2011-0638 and BIO2013-44647-R.Peer reviewedPeer Reviewe

Pea PSII-LHCII supercomplexes form pairs by making connections across the stromal gap

In higher plant thylakoids, the heterogeneous distribution of photosynthetic protein complexes is a determinant for the formation of grana, stacks of membrane discs that are densely populated with Photosystem II (PSII) and its light harvesting complex (LHCII). PSII associates with LHCII to form the PSII-LHCII supercomplex, a crucial component for solar energy conversion. Here, we report a biochemical, structural and functional characterization of pairs of PSII-LHCII supercomplexes, which were isolated under physiologically-relevant cation concentrations. Using single-particle cryo-electron microscopy, we determined the three-dimensional structure of paired C2S2M PSII-LHCII supercomplexes at 14 angstrom resolution. The two supercomplexes interact on their stromal sides through a specific overlap between apposing LHCII trimers and via physical connections that span the stromal gap, one of which is likely formed by interactions between the N-terminal loops of two Lhcb4 monomeric LHCII subunits. Fast chlorophyll fluorescence induction analysis showed that paired PSII-LHCII supercomplexes are energetically coupled. Molecular dynamics simulations revealed that additional flexible physical connections may form between the apposing LHCII trimers of paired PSII-LHCII supercomplexes in appressed thylakoid membranes. Our findings provide new insights into how interactions between pairs of PSII-LHCII supercomplexes can link adjacent thylakoids to mediate the stacking of grana membranes

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

Biophysical characterization of a chaperone complex involved in macroautophagy

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura:31-01-2020Esta tesis tiene embargado el acceso al texto completo hasta el 31-07-2021Eukaryotic cells have developed regulated pathways to maintain a fine balance between protein synthesis, folding, trafficking and degradation (proteostasis). Disruption of this homeostasis network may lead to the onset of pathologies such as cancer or neurodegenerative disorders. Therefore, cells count on several pathways to remove and prevent accumulation and aggregation of potentially toxic proteins, namely the ubiquitin-proteasome system (UPS) and autophagy. Three types of autophagy have been described so far: chaperone-mediated autophagy (CMA), microautophagy and macroautophagy. The chaperone Hsp70 has a central role in all of them. In macroautophagy, Hsp70, in collaboration with other partners (CHIP, Bag3, HspB8), forms a complex that recognizes, ubiquitinates and delivers aggregate-prone proteins towards special locations in cells (aggresomes), for further degradation. This project focuses on the biophysical characterization of the HspB8:Bag3:Hsp70 complex and its components. HspB8, Bag3 and Hsp70 form a stable ternary complex in vitro, which we have studied by different biophysical techniques and also by standard electron microscopy (negative staining) and cryoelectron microscopy (cryoEM). Using cryoelectron tomography, we have analyzed the distribution of the ternary complex in the specimen grids, and have found a tendency of the complex not to distribute evenly within the ice layer, but in a single stratum probably the air-water interface. Using single particle analysis, a low-resolution map has been obtained by negative staining, which combined with crosslinking-mass spectrometry data, has allowed us to build a pseudo-atomic model of the ternary complex. Additionally, we have obtained by cryoEM a low-resolution 3D map where only the atomic models of Hsp70SBD and BAG:Hsp70NBD could be accommodated. The model supports the previously described bimodal character of the Bag3:Hsp70 interaction

Characterization of CRISPR RNA guided immunity in Bacillus halodurans type I-C system

Prokaryotes utilize the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) – Cas (CRISPR-associated) adaptive immune system to defend against infection. A CRISPR locus consists of an AT-rich leader region followed by a series of DNA repeats interspersed by foreign DNA-derived spacers. Upon viral infection, Cas proteins acquire short fragments from the invader and insert them as new spacers into the CRISPR locus. CRISPR transcripts are generated from the CRISPR locus and assemble with Cas proteins to form the surveillance complex. The CRISPR RNA guides the complex to target foreign genetic elements bearing sequence complementarity to the crRNA and recruits a Cas nuclease for degradation. The research presented in this dissertation focuses on understanding the mechanisms of CRISPR RNA guided immunity in Bacillus halodurans type I-C system during adaptation and interference. Cas4 is widespread across types I, II and V and is thought to be involved in spacer acquisition along with the universally conserved Cas1 and Cas2 proteins, but the role of Cas4 has remained unclear. Using a combination of biochemical and structural experiments, we reveal that type I-C Cas4 in B. halodurans interacts directly with Cas1 and Cas2, forming a Cas4-Cas1-Cas2 complex, that mediates spacer selection, processing, and integration during CRISPR immunity. Cas4 associates tightly with Cas1 and the presence of CRISPR DNA substrates helps to stabilize the higher order complex. Cas4 selectively captures spacers that contain protospacer adjacent motifs (PAMs), short sequences required for proper target recognition by the surveillance complex, and processes the substrate directly upstream of the PAM site. When in complex with Cas1-Cas2, Cas4 cleaves spacers endonucleolytically and the complex preferentially integrates the processed spacers at the leader-repeat junction in the CRISPR locus. Together, our findings demonstrate that Cas4 is indispensable in CRISPR immunity by providing functional spacers for target recognition. For target recognition, type I-C system is unique in that only three proteins are required to form its surveillance complex. It is unknown how type I-C Cascade searches for targets using this minimal machinery. We investigated binding interactions of B. halodurans type I-C Cascade with dsDNA and found that, unlike E. coli type I-E Cascade, type I-C Cascade has much strong non-specific affinity for DNA. These observations suggest a search mechanism involving longer-lived interactions with DNA, potentially through one-dimensional sliding. To test this, we initiated development of a single-molecule fluorescence resonance energy transfer (FRET) assay to directly visualize how Cascade searches target DNA in real time. We constructed a system suitable for labeling type I-C Cascade with a fluorophore for the smFRET assay. Using this system, we detected bulk FRET between Cy3-labelled dsDNA target and Cy5-labelled Cascade upon DNA binding. These experiments established a FRET system that will be used for future smFRET experiments to understand the kinetics and mechanisms for searching DNA targets by type I-C Cascade