High-resolution Structural Data Research Articles

PyRBDome: a comprehensive computational platform for enhancing RNA-binding proteome data.

High-throughput proteomics approaches have revolutionised the identification of RNA-binding proteins (RBPome) and RNA-binding sequences (RBDome) across organisms. Yet, the extent of noise, including false positives, associated with these methodologies, is difficult to quantify as experimental approaches for validating the results are generally low throughput. To address this, we introduce pyRBDome, a pipeline for enhancing RNA-binding proteome data in silico. It aligns the experimental results with RNA-binding site (RBS) predictions from distinct machine-learning tools and integrates high-resolution structural data when available. Its statistical evaluation of RBDome data enables quick identification of likely genuine RNA-binders in experimental datasets. Furthermore, by leveraging the pyRBDome results, we have enhanced the sensitivity and specificity of RBS detection through training new ensemble machine-learning models. pyRBDome analysis of a human RBDome dataset, compared with known structural data, revealed that although UV-cross-linked amino acids were more likely to contain predicted RBSs, they infrequently bind RNA in high-resolution structures. This discrepancy underscores the limitations of structural data as benchmarks, positioning pyRBDome as a valuable alternative for increasing confidence in RBDome datasets.

In silico studies of the open form of human tissue transglutaminase

Human tissue transglutaminase (tTG) is an intriguing multifunctional enzyme involved in various diseases, including celiac disease and neurological disorders. Although a number of tTG inhibitors have been developed, the molecular determinants governing ligand binding remain incomplete due to the lack of high-resolution structural data in the vicinity of its active site. In this study, we obtained the complete high-resolution model of tTG by in silico methods based on available PDB structures. We discovered significant differences in the active site architecture between our and known tTG models, revealing an additional loop which affects the ligand binding affinity. We assembled a library of new potential tTG inhibitors based on the obtained complete model of the enzyme. Our library substantially expands the spectrum of possible drug candidates targeting tTG and encompasses twelve molecular scaffolds, eleven of which are novel and exhibit higher binding affinity then already known ones, according to our in silico studies. The results of this study open new directions for structure-based drug design of tTG inhibitors, offering the complete protein model and suggesting a wide range of new compounds for further experimental validation.

Structure analysis of the telomere resolvase from the Lyme disease spirochete Borrelia garinii reveals functional divergence of its C-terminal domain.

Borrelia spirochetes are the causative agents of Lyme disease and relapsing fever, two of the most common tick-borne illnesses. A characteristic feature of these spirochetes is their highly segmented genomes which consists of a linear chromosome and a mixture of up to approximately 24 linear and circular extrachromosomal plasmids. The complexity of this genomic arrangement requires multiple strategies for efficient replication and partitioning during cell division, including the generation of hairpin ends found on linear replicons mediated by the essential enzyme ResT, a telomere resolvase. Using an integrative structural biology approach employing advanced modelling, circular dichroism, X-ray crystallography and small-angle X-ray scattering, we have generated high resolution structural data on ResT from B. garinii. Our data provides the first high-resolution structures of ResT from Borrelia spirochetes and revealed active site positioning in the catalytic domain. We also demonstrate that the C-terminal domain of ResT is required for both transesterification steps of telomere resolution, and is a requirement for DNA binding, distinguishing ResT from other telomere resolvases from phage and bacteria. These results advance our understanding of the molecular function of this essential enzyme involved in genome maintenance in Borrelia pathogens.

A Strep-tag Imprinted Polymer Platform for Heterogenous Bio(electro)catalysis.

Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising-strategies for protein MIPs relies on the exploitation of short surface-exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high-resolution structural data of flexible surface-exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin-based MIP that recognizes recombinant proteins via the widely used Strep-tag II affinity peptide. Electrochemistry, surface-sensitive spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep-MIP electrosynthesis. The functionality of this novel platform was verified with two Strep-tag labeled enzymes: an O2-tolerant [NiFe]-hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep-MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology.

Investigating the neural mechanisms of transcranial direct current stimulation effects on human cognition: current issues and potential solutions.

Transcranial direct current stimulation (tDCS) has been studied extensively for its potential to enhance human cognitive functions in healthy individuals and to treat cognitive impairment in various clinical populations. However, little is known about how tDCS modulates the neural networks supporting cognition and the complex interplay with mediating factors that may explain the frequently observed variability of stimulation effects within and between studies. Moreover, research in this field has been characterized by substantial methodological variability, frequent lack of rigorous experimental control and small sample sizes, thereby limiting the generalizability of findings and translational potential of tDCS. The present manuscript aims to delineate how these important issues can be addressed within a neuroimaging context, to reveal the neural underpinnings, predictors and mediators of tDCS-induced behavioral modulation. We will focus on functional magnetic resonance imaging (fMRI), because it allows the investigation of tDCS effects with excellent spatial precision and sufficient temporal resolution across the entire brain. Moreover, high resolution structural imaging data can be acquired for precise localization of stimulation effects, verification of electrode positions on the scalp and realistic current modeling based on individual head and brain anatomy. However, the general principles outlined in this review will also be applicable to other imaging modalities. Following an introduction to the overall state-of-the-art in this field, we will discuss in more detail the underlying causes of variability in previous tDCS studies. Moreover, we will elaborate on design considerations for tDCS-fMRI studies, optimization of tDCS and imaging protocols and how to assure high-level experimental control. Two additional sections address the pressing need for more systematic investigation of tDCS effects across the healthy human lifespan and implications for tDCS studies in age-associated disease, and potential benefits of establishing large-scale, multidisciplinary consortia for more coordinated tDCS research in the future. We hope that this review will contribute to more coordinated, methodologically sound, transparent and reproducible research in this field. Ultimately, our aim is to facilitate a better understanding of the underlying mechanisms by which tDCS modulates human cognitive functions and more effective and individually tailored translational and clinical applications of this technique in the future.

Formation of Peroxynitrite, [O-N-O-O]-, via a Cascade of Reactions between Ozonide and Ammonia.

We report on an unexpected reaction between ammonia and potassium ozonide dissolved in liq. NH3 resulting in the formation of peroxynitrite, [ONOO]-, which exclusively happens in the presence of a specific partially fluorinated aniline-based ammonium cation. High-resolution structural data of the peroxynitrite anion in cis-conformation have been obtained. We further studied this molecule anion by single crystal Raman spectroscopy. The cis and trans isomers of peroxynitrite were analysed computationally with respect to their relative energies, the cis-trans transition barrier and their decomposition pathways to the nitrate anion. By experimentally examining cations decorated with fluorine ligands to different degrees, we demonstrate that fluorine specific interactions play a crucial role in the unexpected formation of peroxynitrite and as a conspicuously structure directing factor for the underlying crystalline solid phases, exhibiting distinct micro-separations of fluorine and hydrogen enriched regions.

Progressive increase of brain gray matter volume in individuals with regular soccer training

The study aimed to investigate alterations in gray matter volume in individuals undergoing regular soccer training, using high-resolution structural data, while also examining the temporal precedence of such structural alterations. Both voxel-based morphometry and source-based morphometry (SBM) methods were employed to analyze volumetric changes in gray matter between the soccer and control groups. Additionally, a causal network of structural covariance (CaSCN) was built using granger causality analysis on brain structural data ordering by training duration. Significant increases in gray matter volume were observed in the cerebellum in the soccer group. Additionally, the results of the SBM analysis revealed significant increases in gray matter volume in the calcarine and thalamus of the soccer group. The analysis of CaSCN demonstrated that the thalamus had a prominent influence on other brain regions in the soccer group, while the calcarine served as a transitional node, and the cerebellum acted as a prominent node that could be easily influenced by other brain regions. In conclusion, our study identified widely affected regions with increased gray matter volume in individuals with regular soccer training. Furthermore, a temporal precedence relationship among these regions was observed.

Three Biopolymers and Origin of Life Scenarios.

To track down the possible roots of life, various models for the initial living system composed of different combinations of the three extant biopolymers, RNA, DNA, and proteins, are presented. The suitability of each molecular set is assessed according to its ability to emerge autonomously, sustain, and evolve continuously towards life as we know it. The analysis incorporates current biological knowledge gained from high-resolution structural data and large sequence datasets, together with experimental results concerned with RNA replication and with the activity demonstrated by standalone constructs of the ribosomal Peptidyl Transferase Center region. The scrutiny excludes the DNA-protein combination and assigns negligible likelihood to the existence of an RNA-DNA world, as well as to an RNA world that contained a replicase made of RNA. It points to the precedence of an RNA-protein system, whose model of emergence suggests specific processes whereby a coded proto-ribosome ribozyme, specifically aminoacylated proto-tRNAs and a proto-polymerase enzyme, could have autonomously emerged, cross-catalyzing the formation of each other. This molecular set constitutes a feasible starting point for a continuous evolutionary path, proceeding via natural processes from the inanimate matter towards life as we know it.

Characterization of β-Barrel Outer Membrane Proteins and Their Interactions with Chaperones by Chemical-Crosslinking Mass Spectrometry.

Chemical crosslinking-mass spectrometry (XL-MS) is an established tool that can be used to study the architecture and dynamics of proteins and protein assemblies. Here the application of XL-MS to study outer membrane proteins (OMPs) and their interactions with periplasmic chaperones is described, to inform on the molecular mechanisms underpinning OMP assembly. XL-MS data are especially powerful when used to complement high-resolution structural data, data from structural prediction or to drive molecular modeling of proteins and protein assemblies. The approach described here could be applied to the study of any protein assembly (including other membrane proteins).

Structural Insights into the Activation of Human Aryl Hydrocarbon Receptor by the Environmental Contaminant Benzo[a]pyrene and Structurally Related Compounds

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor belonging to the bHLH/PAS protein family and responding to hundreds of natural and chemical substances. It is primarily involved in the defense against chemical insults and bacterial infections or in the adaptive immune response, but also in the development of pathological conditions ranging from inflammatory to neoplastic disorders. Despite its prominent roles in many (patho)physiological processes, the lack of high-resolution structural data has precluded for thirty years an in-depth understanding of the structural mechanisms underlying ligand-binding specificity, promiscuity and activation of AHR. We recently reported a cryogenic electron microscopy (cryo-EM) structure of human AHR bound to the natural ligand indirubin, the chaperone Hsp90 and the co-chaperone XAP2 that provided the first experimental visualization of its ligand-binding PAS-B domain. Here, we report a 2.75 Å resolution structure of the AHR complex bound to the environmental pollutant benzo[a]pyrene (B[a]P). The structure substantiates the existence of a bipartite PAS-B ligand-binding pocket with a geometrically constrained primary binding site controlling ligand binding specificity and affinity, and a secondary binding site contributing to the binding promiscuity of AHR. We also report a docking study of B[a]P congeners that validates the B[a]P-bound PAS-B structure as a suitable model for accurate computational ligand binding assessment. Finally, comparison of our agonist-bound complex with the recently reported structures of mouse and fruit fly AHR PAS-B in different activation states suggests a ligand-induced loop conformational change potentially involved in the regulation of AHR function.

Periodontitis is associated with altered brain structure and function in normal cognition middle-aged and elderly individuals.

Numerous studies have proposed that periodontitis is a potential risk factor for Alzheimer's disease. However, the association between periodontitis and brain normal cognition in aged and elderly individuals (NCs) is unclear. Such a link could provide clues to Alzheimer's disease development and strategies for early prevention. To explore the associations between periodontal condition and metrics of both brain structure and function among NCs with the help of multimodal magnetic resonance imaging (MRI). High-resolution T1-weighted structural data, resting-state functional-MRI data, and measures of periodontal condition were collected from 40 NCs. Cortical volume, thickness, and area as well as regional homogeneity were calculated with the aid of DPABISurf software. Correlation analyses were then conducted between each imaging metric and periodontal index. Consistent negative correlations were observed between severity of periodontitis (mild, moderate, severe) and cortical volume, area, and thickness, not only in brain regions that took charge of primary function but also in brain regions associated with advanced cognition behavior. Among participants with mild attachment loss (AL) and a shallow periodontal pocket depth (PPD), periodontal index was positively correlated with most measures of brain structure and function, while among participants with severe AL and deep PPD, periodontal index was negatively correlated with measures of brain structure and function (all p < .005 for each hemisphere). Our results demonstrate that periodontitis is associated with widespread changes in brain structure and function among middle-aged and elderly adults without signs of cognitive decline, which might be a potential risk factor for brain damage.

Revisiting bacterial cytolethal distending toxin structure and function.

Cytolethal distending toxins (CDTs) are intracellular-acting bacterial genotoxins generated by a diverse group of mucocutaneous human pathogens. CDTs must successfully bind to the plasma membrane of host cells in order to exert their modulatory effects. Maximal toxin activity requires all three toxin subunits, CdtA, CdtB, and CdtC, which, based primarily on high-resolution structural data, are believed to preassemble into a tripartite complex necessary for toxin activity. However, biologically active toxin has not been experimentally demonstrated to require assembly of the three subunits into a heterotrimer. Here, we experimentally compared concentration-dependent subunit interactions and toxin cellular activity of the Campylobacter jejuni CDT (Cj-CDT). Co-immunoprecipitation and dialysis retention experiments provided evidence for the presence of heterotrimeric toxin complexes, but only at concentrations of Cj-CdtA, Cj-CdtB, and Cj-CdtC several logs higher than required for Cj-CDT-mediated arrest of the host cell cycle at the G2/M interface, which is triggered by the endonuclease activity associated with the catalytic Cj-CdtB subunit. Microscale thermophoresis confirmed that Cj-CDT subunit interactions occur with low affinity. Collectively, our data suggest that at the lowest concentrations of toxin sufficient for arrest of cell cycle progression, mixtures of Cj-CdtA, Cj-CdtB, and Cj-CdtC consist primarily of non-interacting, subunit monomers. The lack of congruence between toxin tripartite structure and cellular activity suggests that the widely accepted model that CDTs principally intoxicate host cells as preassembled heterotrimeric structures should be revisited.

Cryo-EM elucidates the uroplakin complex structure within liquid-crystalline lipids in the porcine urothelial membrane

The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this study, we utilized cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex within the porcine AUM. While the global resolution achieved was 3.5 Å, we acknowledge that due to orientation bias, the resolution in the vertical direction was determined to be 6.3 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.

Fortuitous In Vitro Compound Degradation Produces a Tractable Hit against Mycobacterium tuberculosis Dethiobiotin Synthetase: A Cautionary Tale of What Goes In Does Not Always Come Out.

We previously reported potent ligands and inhibitors of Mycobacterium tuberculosis dethiobiotin synthetase (MtDTBS), a promising target for antituberculosis drug development (Schumann et al., ACS Chem Biol. 2021, 16, 2339-2347); here, the unconventional origin of the fragment compound they were derived from is described for the first time. Compound 1 (9b-hydroxy-6b,7,8,9,9a,9b-hexahydrocyclopenta[3,4]cyclobuta[1,2-c]chromen-6(6aH)-one), identified by an in silico fragment screen, was subsequently shown by surface plasmon resonance to have dose-responsive binding (KD = 0.6 mM). Clear electron density was revealed in the DAPA substrate binding pocket when 1 was soaked into MtDTBS crystals, but the density was inconsistent with the structure of 1. Here, we show that the lactone of 1 hydrolyzes to a carboxylic acid (2) under basic conditions, including those of the crystallography soak, with a subsequent ring opening of the component cyclobutane ring forming a cyclopentylacetic acid (3). Crystals soaked directly with authentic 3 produced an electron density that matched that of crystals soaked with presumed 1, confirming the identity of the bound ligand. The synthetic utility of fortuitously formed 3 enabled the subsequent compound development of nanomolar inhibitors. Our findings represent an example of chemical modification within drug discovery assays and demonstrate the value of high-resolution structural data in the fragment hit validation process.

Multi-Scale Feature Residual Feedback Network for Super-Resolution Reconstruction of the Vertical Structure of the Radar Echo

The vertical structure of radar echo is crucial for understanding complex microphysical processes of clouds and precipitation, and for providing essential data support for the study of low-level wind shear and turbulence formation, evolution, and dissipation. Therefore, finding methods to improve the vertical data resolution of the existing radar network is crucial. Existing algorithms for improving image resolution usually focus on increasing the width and height of images. However, improving the vertical data resolution of weather radar requires a focus on improving the elevation angle resolution while maintaining distance resolution. To address this challenge, we propose a network for super-resolution reconstruction of weather radar echo vertical structures. The network is based on a multi-scale residual feedback network (MR-FBN) and uses new multi-scale feature residual blocks (MSRB) to effectively extract and utilize data features at different scales. The feedback network gradually generates the final high-resolution vertical structure data. In addition, we propose an elevation upsampling layer (EUL) specifically for this task, replacing the traditional image subpixel convolution layer. Experimental results show that the proposed method can effectively improve the elevation angle resolution of weather radar echo vertical structure data, providing valuable help for atmospheric detection.

Progressive structural damage in sleep-related hypermotor epilepsy.

This study aimed to explore the alterations in gray matter volume (GMV) based on high-resolution structural data and the temporal precedence of structural alterations in patients with sleep-related hypermotor epilepsy (SHE). After preprocessing of T1 structural images, the voxel-based morphometry and source-based morphometry (SBM) methods were applied in 60 SHE patients and 56 healthy controls to analyze the gray matter volumetric alterations. Furthermore, a causal network of structural covariance (CaSCN) was constructed using Granger causality analysis based on structural data of illness duration ordering to assess the causal impact of structural changes in abnormal gray matter regions. The GMVs of SHE patients were widely reduced, mainly in the bilateral cerebellums, fusiform gyri, the right angular gyrus, the right postcentral gyrus, and the left parahippocampal gyrus. In addition to those regions, the results of the SBM analysis also found decreased GMV in the bilateral frontal lobes, precuneus, and supramarginal gyri. The analysis of CaSCN showed that along with disease progression, the cerebellum was the prominent node that tended to affect other brain regions in SHE patients, while the frontal lobe was the transition node and the supramarginal gyrus was the prominent node that may be easily affected by other brain regions. Our study found widely affected regions of decreased GMVs in SHE patients; these regions underlie the morphological basis of epileptic networks, and there is a temporal precedence relationship between them.

Cryo-EM structures and binding of mouse and human ACE2 to SARS-CoV-2 variants of concern indicate that mutations enabling immune escape could expand host range.

Investigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations. There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected. Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors. Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor.

Structural Biochemistry of Muscle Contraction.

Muscles are essential for movement and heart function. Contraction and relaxation of muscles rely on the sliding of two types of filaments-the thin filament and the thick myosin filament. The thin filament is composed mainly of filamentous actin (F-actin), tropomyosin, and troponin. Additionally, several other proteins are involved in the contraction mechanism, and their malfunction can lead to diverse muscle diseases, such as cardiomyopathies. We review recent high-resolution structural data that explain the mechanism of action of muscle proteins at an unprecedented level of molecular detail. We focus on the molecular structures of the components of the thin and thick filaments and highlight the mechanisms underlying force generation through actin-myosin interactions, as well as Ca2+-dependent regulation via the dihydropyridine receptor, the ryanodine receptor, and troponin. We particularly emphasize the impact of cryo-electron microscopy and cryo-electron tomography in leading muscle research into a new era.

Reorganization of brain networks in patients with temporal lobe epilepsy and comorbid headache

ObjectiveThe white matter structural network changes remain poorly understood in patients with temporal lobe epilepsy and comorbid headache (PWH). This study aimed at exploring topological changes in the structural network. MethodsTwenty-five PWH, 32 patients with temporal lobe epilepsy without headache, and 22 healthy controls were recruited in this study. High-resolution structural MRI and diffusion tensor imaging data were acquired from these participants. A graph theory-based approach was employed to characterize the topological properties of the structural network. A network-based statistical analysis was employed to explore abnormal connectivity alterations in PWH. ResultsCompared with healthy controls, PWH exhibited significantly decreased small-world index, shortest path length, increased clustering coefficient, global efficiency, and local efficiency. Patients with temporal lobe epilepsy and comorbid headache displayed a significantly reduced small-world index, shortest path length, and increased global efficiency when compared with patients with temporal lobe epilepsy without headache. In addition, PWH exhibited abnormal local network parameters, mainly located in the prefrontal, temporal, occipital, and parietal regions. Furthermore, network-based statistical analysis revealed that PWH had abnormal structural connections between the temporoparietal lobe, occipital lobe, insula, cingulate gyrus, and thalamus. ConclusionThis study reveals the abnormal white matter structural network alterations in PWH, allowing a better insight into the neuroanatomical mechanisms that predispose epileptic patients to comorbid headaches from the network levels.

Recent advancements in the structural biology of human telomerase and their implications for improved design of cancer therapeutics.

Telomerase is a specialized reverse transcriptase that synthesizes telomeric repeats at the ends of linear chromosomes. Telomerase is transiently expressed in germ and stem cells, but nearly all somatic cells silence it after differentiating. However, the vast majority of cancer cells reactivate and constitutively express telomerase to maintain replicative immortality. Because of this, telomerase has remained a promising broad-spectrum chemotherapeutic target for over 30 years. However, various challenges associated with obtaining high-resolution structural data for telomerase have limited the development of rationally designed structure-based therapeutics. Various techniques and model systems have been utilized to advance our understanding of the structural biology of telomerase. In particular, multiple high-resolution cryogenic electron microscopy (cryo-EM) structures published within the past few years have revealed new components of the telomerase complex with near atomic resolution structural models. Additionally, these structures have provided details for how telomerase is recruited to telomeres and its mechanism of telomere synthesis. With these new pieces of evidence, and the promising outlook for future refinements to our current models, the possibility of telomerase specific chemotherapeutics is becoming more tangible than ever. This review summarizes these recent advancements and outlines outstanding questions in the field.