Dissecting the Conformational Heterogeneity of Stem-Loop Substructures of the Fifth Element in the 5'-Untranslated Region of SARS-CoV-2.

Throughout the family of coronaviruses, structured RNA elements within the 5' region of the genome are highly conserved. The fifth stem-loop element from SARS-CoV-2 (5_SL5) represents an example of an RNA structural element, repeatedly occurring in coronaviruses. It contains a conserved, repetitive fold within its substructures SL5a and SL5b. We herein report the detailed characterization of the structure and dynamics of elements SL5a and SL5b that are located immediately upstream of the SARS-CoV-2 ORF1a/b start codon. Exploiting the unique ability of solution NMR methods, we show that the structures of both apical loops are modulated by structural differences in the remote parts located in their stem regions. We further integrated our high-resolution models of SL5a/b into the context of full-length 5_SL5 structures by combining different structural biology methods. Finally, we evaluated the impact of the two most common VoC mutations within 5_SL5 with respect to individual base-pair stability.

Correction: The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: The case of the spike A222V mutation.

[This corrects the article DOI: 10.1371/journal.ppat.1010631.].

The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: The case of the spike A222V mutation.

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has since reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness.

Emerging Themes in CryoEM─Single Particle Analysis Image Processing.

Cryo-electron microscopy (CryoEM) has become a vital technique in structural biology. It is an interdisciplinary field that takes advantage of advances in biochemistry, physics, and image processing, among other disciplines. Innovations in these three basic pillars have contributed to the boosting of CryoEM in the past decade. This work reviews the main contributions in image processing to the current reconstruction workflow of single particle analysis (SPA) by CryoEM. Our review emphasizes the time evolution of the algorithms across the different steps of the workflow differentiating between two groups of approaches: analytical methods and deep learning algorithms. We present an analysis of the current state of the art. Finally, we discuss the emerging problems and challenges still to be addressed in the evolution of CryoEM image processing methods in SPA.

Structural analysis of receptors and actin polarity in platelet protrusions.

During activation the platelet cytoskeleton is reorganized, inducing adhesion to the extracellular matrix and cell spreading. These processes are critical for wound healing and clot formation. Initially, this task relies on the formation of strong cellular-extracellular matrix interactions, exposed in subendothelial lesions. Despite the medical relevance of these processes, there is a lack of high-resolution structural information on the platelet cytoskeleton controlling cell spreading and adhesion. Here, we present in situ structural analysis of membrane receptors and the underlying cytoskeleton in platelet protrusions by applying cryoelectron tomography to intact platelets. We utilized three-dimensional averaging procedures to study receptors at the plasma membrane. Analysis of substrate interaction-free receptors yielded one main structural class resolved to 26 Å, resembling the αIIbß3 integrin folded conformation. Furthermore, structural analysis of the actin network in pseudopodia indicates a nonuniform polarity of filaments. This organization would allow generation of the contractile forces required for integrin-mediated cell adhesion.

Scipion-EM-ProDy: A Graphical Interface for the ProDy Python Package within the Scipion Workflow Engine Enabling Integration of Databases, Simulations and Cryo-Electron Microscopy Image Processing.

Macromolecular assemblies, such as protein complexes, undergo continuous structural dynamics, including global reconfigurations critical for their function. Two fast analytical methods are widely used to study these global dynamics, namely elastic network model normal mode analysis and principal component analysis of ensembles of structures. These approaches have found wide use in various computational studies, driving the development of complex pipelines in several software packages. One common theme has been conformational sampling through hybrid simulations incorporating all-atom molecular dynamics and global modes of motion. However, wide functionality is only available for experienced programmers with limited capabilities for other users. We have, therefore, integrated one popular and extensively developed software for such analyses, the ProDy Python application programming interface, into the Scipion workflow engine. This enables a wider range of users to access a complete range of macromolecular dynamics pipelines beyond the core functionalities available in its command-line applications and the normal mode wizard in VMD. The new protocols and pipelines can be further expanded and integrated into larger workflows, together with other software packages for cryo-electron microscopy image analysis and molecular simulations. We present the resulting plugin, Scipion-EM-ProDy, in detail, highlighting the rich functionality made available by its development.

3DBionotes COVID-19 edition.

SUMMARY: The web platform 3DBionotes-WS integrates multiple web services and an interactive web viewer to provide a unified environment in which biological annotations can be analyzed in their structural context. Since the COVID-19 outbreak, new structural data from many viral proteins have been provided at a very fast pace. This effort includes many cryogenic electron microscopy (cryo-EM) studies, together with more traditional ones (X-rays, NMR), using several modeling approaches and complemented with structural predictions. At the same time, a plethora of new genomics and interactomics information (including fragment screening and structure-based virtual screening efforts) have been made available from different servers. In this context, we have developed 3DBionotes-COVID-19 as an answer to: (i) the need to explore multiomics data in a unified context with a special focus on structural information and (ii) the drive to incorporate quality measurements, especially in the form of advanced validation metrics for cryo-EM. AVAILABILITY AND IMPLEMENTATION: https://3dbionotes.cnb.csic.es/ws/covid19. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Identification of incorrectly oriented particles in cryo-EM single particle analysis.

The quality of a 3D map produced by the single-particle analysis method is highly dependent on an accurate assignment of orientations to the many experimental images. However, the problem's complexity implies the presence of several local minima in the optimized goal functions. Consequently, validation methods to confirm the angular assignment are very useful to yield higher-resolution 3D maps. In this work, we present a graph-signal-processing-based methodology that analyzes the correlation landscape as a function of the orientation, an approach allowing the estimation of the assigned orientations' reliability. Using this method, we may identify low-reliability images that probably incorrectly contribute to the final 3D reconstruction.

Automatic local resolution-based sharpening of cryo-EM maps.

MOTIVATION: Recent technological advances and computational developments have allowed the reconstruction of Cryo-Electron Microscopy (cryo-EM) maps at near-atomic resolution. On a typical workflow and once the cryo-EM map has been calculated, a sharpening process is usually performed to enhance map visualization, a step that has proven very important in the key task of structural modeling. However, sharpening approaches, in general, neglects the local quality of the map, which is clearly suboptimal. RESULTS: Here, a new method for local sharpening of cryo-EM density maps is proposed. The algorithm, named LocalDeblur, is based on a local resolution-guided Wiener restoration approach of the original map. The method is fully automatic and, from the user point of view, virtually parameter-free, without requiring either a starting model or introducing any additional structure factor correction or boosting. Results clearly show a significant impact on map interpretability, greatly helping modeling. In particular, this local sharpening approach is especially suitable for maps that present a broad resolution range, as is often the case for membrane proteins or macromolecules with high flexibility, all of them otherwise very suitable and interesting specimens for cryo-EM. To our knowledge, and leaving out the use of local filters, it represents the first application of local resolution in cryo-EM sharpening. AVAILABILITY AND IMPLEMENTATION: The source code (LocalDeblur) can be found at https://github.com/I2PC/xmipp and can be run using Scipion (http://scipion.cnb.csic.es) (release numbers greater than or equal 1.2.1). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The first single particle analysis Map Challenge: A summary of the assessments.

The recent successes of cryo-electron microscopy fostered great expectation of solving many new and previously recalcitrant biomolecular structures. However, it also brings with it the danger of compromising the validity of the outcomes if not done properly. The Map Challenge is a first step in assessing the state of the art and to shape future developments in data processing. The organizers presented seven cases for single particle reconstruction, and 27 members of the community responded with 66 submissions. Seven groups analyzed these submissions, resulting in several assessment reports, summarized here. We devised a range of analyses to evaluate the submitted maps, including visual impressions, Fourier shell correlation, pairwise similarity and interpretation through modeling. Unfortunately, we did not find strong trends. We ascribe this to the complexity of the challenge, dealing with multiple cases, software packages and processing approaches. This puts the user in the spotlight, where his/her choices becomes the determinant of map quality. The future focus should therefore be on promulgating best practices and encapsulating these in the software. Such practices include adherence to validation principles, most notably the processing of independent sets, proper resolution-limited alignment, appropriate masking and map sharpening. We consider the Map Challenge to be a highly valuable exercise that should be repeated frequently or on an ongoing basis.

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.

3DCONS-DB: A Database of Position-Specific Scoring Matrices in Protein Structures.

Many studies have used position-specific scoring matrices (PSSM) profiles to characterize residues in protein structures and to predict a broad range of protein features. Moreover, PSSM profiles of Protein Data Bank (PDB) entries have been recalculated in many works for different purposes. Although the computational cost of calculating a single PSSM profile is affordable, many statistical studies or machine learning-based methods used thousands of profiles to achieve their goals, thereby leading to a substantial increase of the computational cost. In this work we present a new database compiling PSSM profiles for the proteins of the PDB. Currently, the database contains 333,532 protein chain profiles involving 123,135 different PDB entries.

3DIANA: 3D Domain Interaction Analysis: A Toolbox for Quaternary Structure Modeling.

Electron microscopy (EM) is experiencing a revolution with the advent of a new generation of Direct Electron Detectors, enabling a broad range of large and flexible structures to be resolved well below 1 nm resolution. Although EM techniques are evolving to the point of directly obtaining structural data at near-atomic resolution, for many molecules the attainable resolution might not be enough to propose high-resolution structural models. However, accessing information on atomic coordinates is a necessary step toward a deeper understanding of the molecular mechanisms that allow proteins to perform specific tasks. For that reason, methods for the integration of EM three-dimensional maps with x-ray and NMR structural data are being developed, a modeling task that is normally referred to as fitting, resulting in the so called hybrid models. In this work, we present a novel application-3DIANA-specially targeted to those cases in which the EM map resolution is medium or low and additional experimental structural information is scarce or even lacking. In this way, 3DIANA statistically evaluates proposed/potential contacts between protein domains, presents a complete catalog of both structurally resolved and predicted interacting regions involving these domains and, finally, suggests structural templates to model the interaction between them. The evaluation of the proposed interactions is computed with DIMERO, a new method that scores physical binding sites based on the topology of protein interaction networks, which has recently shown the capability to increase by 200% the number of domain-domain interactions predicted in interactomes as compared to previous approaches. The new application displays the information at a sequence and structural level and is accessible through a web browser or as a Chimera plugin at http://3diana.cnb.csic.es.

StructMap: Elastic Distance Analysis of Electron Microscopy Maps for Studying Conformational Changes.

Single-particle electron microscopy (EM) has been shown to be very powerful for studying structures and associated conformational changes of macromolecular complexes. In the context of analyzing conformational changes of complexes, distinct EM density maps obtained by image analysis and three-dimensional (3D) reconstruction are usually analyzed in 3D for interpretation of structural differences. However, graphic visualization of these differences based on a quantitative analysis of elastic transformations (deformations) among density maps has not been done yet due to a lack of appropriate methods. Here, we present an approach that allows such visualization. This approach is based on statistical analysis of distances among elastically aligned pairs of EM maps (one map is deformed to fit the other map), and results in visualizing EM maps as points in a lower-dimensional distance space. The distances among points in the new space can be analyzed in terms of clusters or trajectories of points related to potential conformational changes. The results of the method are shown with synthetic and experimental EM maps at different resolutions.

Alignment of direct detection device micrographs using a robust Optical Flow approach.

The introduction of direct detection devices in cryo-EM has shown that specimens present beam-induced motion (BIM). Consequently, in this work, we develop a BIM correction method at the image level, resulting in an integrated image in which the in-plane BIM blurring is compensated prior to particle picking. The methodology is based on a robust Optical Flow (OF) approach that can efficiently correct for local movements in a rapid manner. The OF works particularly well if the BIM pattern presents a substantial degree of local movements, which occurs in our data sets for Falcon II data. However, for those cases in which the BIM pattern corresponds to global movements, we have found it advantageous to first run a global motion correction approach and to subsequently apply OF. Additionally, spatial analysis of the Optical Flow allows for quantitative analysis of the BIM pattern. The software that incorporates the new approach is available in XMIPP (http://xmipp.cnb.csic.es).

Electron microscopy studies on the quaternary structure of p53 reveal different binding modes for p53 tetramers in complex with DNA.

The multidomain homotetrameric tumor suppressor p53 has two modes of binding dsDNA that are thought to be responsible for scanning and recognizing specific response elements (REs). The C termini bind nonspecifically to dsDNA. The four DNA-binding domains (DBDs) bind REs that have two symmetric 10 base-pair sequences. p53 bound to a 20-bp RE has the DBDs enveloping the DNA, which is in the center of the molecule surrounded by linker sequences to the tetramerization domain (Tet). We investigated by electron microscopy structures of p53 bound to DNA sequences consisting of a 20-bp RE with either 12 or 20 bp nonspecific extensions on either end. We found a variety of structures that give clues to recognition and scanning mechanisms. The 44- and 60-bp sequences gave rise to three and four classes of structures, respectively. One was similar to the known 20-bp structure, but the DBDs in the other classes were loosely arranged and incompatible with specific DNA recognition. Some of the complexes had density consistent with the C termini extending from Tet to the DNA, adjacent to the DBDs. Single-molecule fluorescence resonance energy transfer experiments detected the approach of the C termini towards the DBDs on addition of DNA. The structural data are consistent with p53 sliding along DNA via its C termini and the DNA-binding domains hopping on and off during searches for REs. The loose structures and posttranslational modifications account for the affinity of nonspecific DNA for p53 and point to a mechanism of enhancement of specificity by its binding to effector proteins.

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

The future of integrated structural biology.

Instruct-ERIC, "the European Research Infrastructure Consortium for Structural biology research," is a pan-European distributed research infrastructure making high-end technologies and methods in structural biology available to users. Here, we describe the current state-of-the-art of integrated structural biology and discuss potential future scientific developments as an impulse for the scientific community, many of which are located in Europe and are associated with Instruct. We reflect on where to focus scientific and technological initiatives within the distributed Instruct research infrastructure. This review does not intend to make recommendations on funding requirements or initiatives directly, neither at the national nor the European level. However, it addresses future challenges and opportunities for the field, and foresees the need for a stronger coordination within the European and international research field of integrated structural biology to be able to respond timely to thematic topics that are often prioritized by calls for funding addressing societal needs.

On the development of three new tools for organizing and sharing information in three-dimensional electron microscopy.

Electron microscopy is a valuable tool for elucidating the three-dimensional structures of macromolecular complexes. As the field matures and the number of solved structures increases, the existence of infrastructures that keep this information organized and accessible is crucial. At the same time, standards and clearly described conventions facilitate software maintenance, benefit interoperability with other packages and allow data interchange. This work describes three developments promoting integrative biology, standardization and workflow processing, namely PeppeR, the EMX initiative and Scipion.

Integrating human and murine anatomical gene expression data for improved comparisons.

MOTIVATION: Information concerning the gene expression pattern in four dimensions (species, genes, anatomy and developmental stage) is crucial for unraveling the roles of genes through time. There are a variety of anatomical gene expression databases, but extracting information from them can be hampered by their diversity and heterogeneity. RESULTS: aGEM 3.1 (anatomic Gene Expression Mapping) addresses the issues of diversity and heterogeneity of anatomical gene expression databases by integrating six mouse gene expression resources (EMAGE, GXD, GENSAT, Allen Brain Atlas data base, EUREXPRESS and BioGPS) and three human gene expression databases (HUDSEN, Human Protein Atlas and BioGPS). Furthermore, aGEM 3.1 provides new cross analysis tools to bridge these resources. AVAILABILITY AND IMPLEMENTATION: aGEM 3.1 can be queried using gene and anatomical structure. Output information is presented in a friendly format, allowing the user to display expression maps and correlation matrices for a gene or structure during development. An in-depth study of a specific developmental stage is also possible using heatmaps that relate gene expression with anatomical components. http://agem.cnb.csic.es CONTACT: natalia@cnb.csic.es SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.