Integrative Structure Modeling: Overview and Assessment.

Integrative structure modeling computationally combines data from multiple sources of information with the aim of obtaining structural insights that are not revealed by any single approach alone. In the first part of this review, we survey the commonly used sources of structural information and the computational aspects of model building. Throughout the past decade, integrative modeling was applied to various biological systems, with a focus on large protein complexes. Recent progress in the field of cryo-electron microscopy (cryo-EM) has resolved many of these complexes to near-atomic resolution. In the second part of this review, we compare a range of published integrative models with their higher-resolution counterparts with the aim of critically assessing their accuracy. This comparison gives a favorable view of integrative modeling and demonstrates its ability to yield accurate and informative results. We discuss possible roles of integrative modeling in the new era of cryo-EM and highlight future challenges and directions.

Regulatory Expansion in Mammals of Multivalent hnRNP Assemblies that Globally Control Alternative Splicing.

Alternative splicing (AS) patterns have diverged rapidly during vertebrate evolution, yet the functions of most species- and lineage-specific splicing events are not known. We observe that mammalian-specific AS events are enriched in transcript sequences encoding intrinsically disordered regions (IDRs) of proteins, in particular those containing glycine/tyrosine repeats that mediate formation of higher-order protein assemblies implicated in gene regulation and human disease. These evolutionary changes impact nearly all members of the hnRNP A and D families of RNA binding proteins. Regulation of these events requires formation of unusual, long-range mammalian-specific RNA duplexes. Differential inclusion of the alternative exons controls the formation of tyrosine-dependent multivalent hnRNP assemblies that, in turn, function to globally regulate splicing. Together, our results demonstrate that AS control of IDR-mediated interactions between hnRNPs represents an important and recurring mechanism underlying splicing regulation. Furthermore, this mechanism has expanded the regulatory capacity of mammalian cells.

Assembly and function of the amyloid-like translational repressor Rim4 is coupled with nutrient conditions.

Amyloid-like protein assemblies have been associated with toxic phenotypes because of their repetitive and stable structure. However, evidence that cells exploit these structures to control function and activity of some proteins in response to stimuli has questioned this paradigm. How amyloid-like assembly can confer emergent functions and how cells couple assembly with environmental conditions remains unclear. Here, we study Rim4, an RNA-binding protein that forms translation-repressing assemblies during yeast meiosis. We demonstrate that in its assembled and repressive state, Rim4 binds RNA more efficiently than in its monomeric and idle state, revealing a causal connection between assembly and function. The Rim4-binding site location within the transcript dictates whether the assemblies can repress translation, underscoring the importance of the architecture of this RNA-protein structure for function. Rim4 assembly depends exclusively on its intrinsically disordered region and is prevented by the Ras/protein kinase A signaling pathway, which promotes growth and suppresses meiotic entry in yeast. Our results suggest a mechanism whereby cells couple a functional protein assembly with a stimulus to enforce a cell fate decision.

The mechanics of correlated variability in segregated cortical excitatory subnetworks.

Understanding the genesis of shared trial-to-trial variability in neuronal population activity within the sensory cortex is critical to uncovering the biological basis of information processing in the brain. Shared variability is often a reflection of the structure of cortical connectivity since it likely arises, in part, from local circuit inputs. A series of experiments from segregated networks of (excitatory) pyramidal neurons in the mouse primary visual cortex challenge this view. Specifically, the across-network correlations were found to be larger than predicted given the known weak cross-network connectivity. We aim to uncover the circuit mechanisms responsible for these enhanced correlations through biologically motivated cortical circuit models. Our central finding is that coupling each excitatory subpopulation with a specific inhibitory subpopulation provides the most robust network-intrinsic solution in shaping these enhanced correlations. This result argues for the existence of excitatory-inhibitory functional assemblies in early sensory areas which mirror not just response properties but also connectivity between pyramidal cells. Furthermore, our findings provide theoretical support for recent experimental observations showing that cortical inhibition forms structural and functional subnetworks with excitatory cells, in contrast to the classical view that inhibition is a nonspecific blanket suppression of local excitation.

A Systems-Level Study Reveals Regulators of Membrane-less Organelles in Human Cells.

Membrane-less organelles (MLOs) are liquid-like subcellular compartments that form through phase separation of proteins and RNA. While their biophysical properties are increasingly understood, their regulation and the consequences of perturbed MLO states for cell physiology are less clear. To study the regulatory networks, we targeted 1,354 human genes and screened for morphological changes of nucleoli, Cajal bodies, splicing speckles, PML nuclear bodies (PML-NBs), cytoplasmic processing bodies, and stress granules. By multivariate analysis of MLO features we identified hundreds of genes that control MLO homeostasis. We discovered regulatory crosstalk between MLOs, and mapped hierarchical interactions between aberrant MLO states and cellular properties. We provide evidence that perturbation of pre-mRNA splicing results in stress granule formation and reveal that PML-NB abundance influences DNA replication rates and that PML-NBs are in turn controlled by HIP kinases. Together, our comprehensive dataset is an unprecedented resource for deciphering the regulation and biological functions of MLOs.

Lateral interactions govern self-assembly of the bacterial biofilm matrix protein BslA.

The soil bacterium Bacillus subtilis is a model organism to investigate the formation of biofilms, the predominant form of microbial life. The secreted protein BslA self-assembles at the surface of the biofilm to give the B. subtilis biofilm its characteristic hydrophobicity. To understand the mechanism of BslA self-assembly at interfaces, here we built a molecular model based on the previous BslA crystal structure and the crystal structure of the BslA paralogue YweA that we determined. Our analysis revealed two conserved protein-protein interaction interfaces supporting BslA self-assembly into an infinite 2-dimensional lattice that fits previously determined transmission microscopy images. Molecular dynamics simulations and in vitro protein assays further support our model of BslA elastic film formation, while mutagenesis experiments highlight the importance of the identified interactions for biofilm structure. Based on this knowledge, YweA was engineered to form more stable elastic films and rescue biofilm structure in bslA deficient strains. These findings shed light on protein film assembly and will inform the development of BslA technologies which range from surface coatings to emulsions in fast-moving consumer goods.

HMMPolish: a coding region polishing tool for TGS-sequenced RNA viruses.

Access to accurate viral genomes is important to downstream data analysis. Third-generation sequencing (TGS) has recently become a popular platform for virus sequencing because of its long read length. However, its per-base error rate, which is higher than next-generation sequencing, can lead to genomes with errors. Polishing tools are thus needed to correct errors either before or after sequence assembly. Despite promising results of available polishing tools, there is still room to improve the error correction performance to perform more accurate genome assembly. The errors, particularly those in coding regions, can hamper analysis such as linage identification and variant monitoring. In this work, we developed a novel pipeline, HMMPolish, for correcting (polishing) errors in protein-coding regions of known RNA viruses. This tool can be applied to either raw TGS reads or the assembled sequences of the target virus. By utilizing profile Hidden Markov Models of protein families/domains in known viruses, HMMPolish can correct errors that are ignored by available polishers. We extensively validated HMMPolish on 34 datasets that covered four clinically important viruses, including HIV-1, influenza-A, norovirus, and severe acute respiratory syndrome coronavirus 2. These datasets contain reads with different properties, such as sequencing depth and platforms (PacBio or Nanopore). The benchmark results against popular/representative polishers show that HMMPolish competes favorably on error correction in coding regions of known RNA viruses.

The Stress Granule Transcriptome Reveals Principles of mRNA Accumulation in Stress Granules.

Stress granules are mRNA-protein assemblies formed from nontranslating mRNAs. Stress granules are important in the stress response and may contribute to some degenerative diseases. Here, we describe the stress granule transcriptome of yeast and mammalian cells through RNA-sequencing (RNA-seq) analysis of purified stress granule cores and single-molecule fluorescence in situ hybridization (smFISH) validation. While essentially every mRNA, and some noncoding RNAs (ncRNAs), can be targeted to stress granules, the targeting efficiency varies from <1% to >95%. mRNA accumulation in stress granules correlates with longer coding and UTR regions and poor translatability. Quantifying the RNA-seq analysis by smFISH reveals that only 10% of bulk mRNA molecules accumulate in mammalian stress granules and that only 185 genes have more than 50% of their mRNA molecules in stress granules. These results suggest that stress granules may not represent a specific biological program of messenger ribonucleoprotein (mRNP) assembly, but instead form by condensation of nontranslating mRNPs in proportion to their length and lack of association with ribosomes.

Atomic structure of Lanreotide nanotubes revealed by cryo-EM.

Functional and versatile nano- and microassemblies formed by biological molecules are found at all levels of life, from cell organelles to full organisms. Understanding the chemical and physicochemical determinants guiding the formation of these assemblies is crucial not only to understand the biological processes they carry out but also to mimic nature. Among the synthetic peptides forming well-defined nanostructures, the octapeptide Lanreotide has been considered one of the best characterized, in terms of both the atomic structure and its self-assembly process. In the present work, we determined the atomic structure of Lanreotide nanotubes at 2.5-Å resolution by cryoelectron microscopy (cryo-EM). Surprisingly, the asymmetric unit in the nanotube contains eight copies of the peptide, forming two tetramers. There are thus eight different environments for the peptide, and eight different conformations in the nanotube. The structure built from the cryo-EM map is strikingly different from the molecular model, largely based on X-ray fiber diffraction, proposed 20 y ago. Comparison of the nanotube with a crystal structure at 0.83-Å resolution of a Lanreotide derivative highlights the polymorphism for this peptide family. This work shows once again that higher-order assemblies formed by even well-characterized small peptides are very difficult to predict.

On the formation of ordered protein assemblies in cell-cell interfaces.

Crystal structures of many cell-cell adhesion receptors reveal the formation of linear "molecular zippers" comprising an ordered one-dimensional array of proteins that form both intercellular (trans) and intracellular (cis) interactions. The clustered protocadherins (cPcdhs) provide an exemplar of this phenomenon and use it as a basis of barcoding of vertebrate neurons. Here, we report both Metropolis and kinetic Monte Carlo simulations of cPcdh zipper formation using simplified models of cPcdhs that nevertheless capture essential features of their three-dimensional structure. The simulations reveal that the formation of long zippers is an implicit feature of cPcdh structure and is driven by their cis and trans interactions that have been quantitatively characterized in previous work. Moreover, in agreement with cryo-electron tomography studies, the zippers are found to organize into two-dimensional arrays even in the absence of attractive interactions between individual zippers. Our results suggest that the formation of ordered two-dimensional arrays of linear zippers of adhesion proteins is a common feature of cell-cell interfaces. From the perspective of simulations, they demonstrate the importance of a realistic depiction of adhesion protein structure and interactions if important biological phenomena are to be properly captured.

Welded Gold Nanoparticle Assemblies Defined Plasmonic Coupling.

Nanoparticle assemblies with interparticle ohmic contacts are crucial for nanodevice fabrication. Despite tremendous progress in DNA-programmable nanoparticle assemblies, seamlessly welding discrete components into welded continuous three-dimensional (3D) configurations remains challenging. Here, we introduce a single-stranded DNA-encoded strategy to customize welded metal nanostructures with tunable morphologies and plasmonic properties. We demonstrate the precise welding of gold nanoparticle assemblies into continuous metal nanostructures with interparticle ohmic contacts through chemical welding in solution. We find that the welded gold nanoparticle assemblies show a consistent morphology with welded efficiency over 90%, such as the rod-like, triangular, and tetrahedral metal nanostructures. Next, we show the versatility of this strategy by welding gold nanoparticle assemblies of varied sizes and shapes. Furthermore, the experiment and simulation show that the welded gold nanoparticle assemblies exhibit defined plasmonic coupling. This single-stranded DNA encoded welding system may provide a new route for accurately building functional plasmonic nanomaterials and devices.

DNA Framework-Programmed Nanoscale Enzyme Assemblies.

Multienzyme assemblies mediated by multivalent interaction play a crucial role in cellular processes. However, the three-dimensional (3D) programming of an enzyme complex with defined enzyme activity in vitro remains unexplored, primarily owing to limitations in precisely controlling the spatial topological configuration. Herein, we introduce a nanoscale 3D enzyme assembly using a tetrahedral DNA framework (TDF), enabling the replication of spatial topological configuration and maintenance of an identical edge-to-edge distance akin to natural enzymes. Our results demonstrate that 3D nanoscale enzyme assemblies in both two-enzyme systems (glucose oxidase (GOx)/horseradish peroxidase (HRP)) and three-enzyme systems (amylglucosidase (AGO)/GOx/HRP) lead to enhanced cascade catalytic activity compared to the low-dimensional structure, resulting in ∼5.9- and ∼7.7-fold enhancements over homogeneous diffusional mixtures of free enzymes, respectively. Furthermore, we demonstrate the enzyme assemblies for the detection of the metabolism biomarkers creatinine and creatine, achieving a low limit of detection, high sensitivity, and broad detection range.

fmr1 Mutation Alters the Early Development of Sensory Coding and Hunting and Social Behaviors in Larval Zebrafish.

Autism spectrum disorders (ASDs) are developmental in origin; however, little is known about how they affect the early development of behavior and sensory coding. The most common inherited form of autism is Fragile X syndrome (FXS), caused by a mutation in FMR1 Mutation of fmr1 in zebrafish causes anxiety-like behavior, hyperactivity, and hypersensitivity in auditory and visual processing. Here, we show that zebrafish fmr1-/- mutant larvae of either sex also display changes in hunting behavior, tectal coding, and social interaction. During hunting, they were less successful at catching prey and displayed altered behavioral sequences. In the tectum, representations of prey-like stimuli were more diffuse and had higher dimensionality. In a social behavioral assay, they spent more time observing a conspecific but responded more slowly to social cues. However, when given a choice of rearing environment fmr1-/- larvae preferred one with reduced visual stimulation, and rearing them in this environment reduced genotype-specific effects on tectal excitability. Together, these results shed new light on how fmr1-/- changes the early development of neural systems and behavior in a vertebrate.SIGNIFICANCE STATEMENT Autism spectrum disorders (ASDs) are caused by changes in early neural development. Animal models of ASDs offer the opportunity to study these developmental processes in greater detail than in humans. Here, we found that a zebrafish mutant for a gene which in humans causes one type of ASD showed early alterations in hunting behavior, social behavior, and how visual stimuli are represented in the brain. However, we also found that mutant fish preferred reduced visual stimulation, and rearing them in this environment reduced alterations in neural activity patterns. These results suggest interesting new directions for using zebrafish as a model to study the development of brain and behavior in ASDs, and how the impact of ASDs could potentially be reduced.

Revolutionizing soybean genomics: How CRISPR and advanced sequencing are unlocking new potential.

Soybean Glycine max L., paleopolyploid genome, poses challenges to its genetic improvement. However, the development of reference genome assemblies and genome sequencing has completely changed the field of soybean genomics, allowing for more accurate and successful breeding techniques as well as research. During the single-cell revolution, one of the most advanced sequencing tools for examining the transcriptome landscape is single-cell RNA sequencing (scRNA-seq). Comprehensive resources for genetic improvement of soybeans may be found in the SoyBase and other genomics databases. CRISPR-Cas9 genome editing technology provides promising prospects for precise genetic modifications in soybean. This method has enhanced several soybean traits, including as yield, nutritional value, and resistance to both biotic and abiotic stresses. With base editing techniques that allow for precise DNA modifications, the use of CRISPR-Cas9 is further increased. With the availability of the reference genome for soybeans and the following assembly of wild and cultivated soybeans, significant chromosomal rearrangements and gene duplication events have been identified, offering new perspectives on the complex genomic structure of soybeans. Furthermore, major single nucleotide polymorphisms (SNPs) linked to stachyose and sucrose content have been found through genome-wide association studies (GWAS), providing important tools for enhancing soybean carbohydrate profiles. In order to open up new avenues for soybean genetic improvement, future research approaches include investigating transcriptional divergence processes, enhancing genetic resources, and incorporating CRISPR-Cas9 technologies.

Genome and transcriptome based comparative analysis of Tilletia indica to decipher the causal genes for pathogenicity of Karnal bunt in wheat.

Tilletia indica Mitra causes Karnal bunt (KB) in wheat by pathogenic dikaryophase. The present study is the first to provide the draft genomes of the dikaryon (PSWKBGD-3) and its two monosporidial lines (PSWKBGH-1 and 2) using Illumina and PacBio reads, their annotation and the comparative analyses among the three genomes by extracting polymorphic SSR markers. The trancriptome from infected wheat grains of the susceptible wheat cultivar WL711 at 24 h, 48h, and 7d after inoculation of PSWKBGH-1, 2 and PSWKBGD-3 were also isolated. Further, two transcriptome analyses were performed utilizing T. indica transcriptome to extract dikaryon genes responsible for pathogenesis, and wheat transcriptome to extract wheat genes affected by dikaryon involved in plant-pathogen interaction during progression of KB in wheat. A total of 54, 529, and 87 genes at 24hai, 48hai, and 7dai, respectively were upregulated in dikaryon stage while 21, 35, and 134 genes of T. indica at 24hai, 48hai, and 7dai, respectively, were activated only in dikaryon stage. While, a total of 23, 17, and 52 wheat genes at 24hai, 48hai, and 7dai, respectively were upregulated due to the presence of dikaryon stage only. The results obtained during this study have been compiled in a web resource called TiGeR ( http://backlin.cabgrid.res.in/tiger/ ), which is the first genomic resource for T. indica cataloguing genes, genomic and polymorphic SSRs of the three T. indica lines, wheat and T. indica DEGs as well as wheat genes affected by T. indica dikaryon along with the pathogenecity related proteins of T. indica dikaryon during incidence of KB at different time points. The present study would be helpful to understand the role of dikaryon in plant-pathogen interaction during progression of KB, which would be helpful to manage KB in wheat, and to develop KB-resistant wheat varieties.

Light and Heat-Driven Flexible Solid Supramolecular Polymer Displaying Phosphorescence and Reversible Photochromism.

Herein, a type of light- and heat-driven flexible supramolecular polymer with reversibly long-lived phosphorescence and photochromism is constructed from acrylamide copolymers with 4-phenylpyridinium derivatives containing a cyano group (P-CN, P-oM, P-mM), sulfobutylether-ß-cyclodextrin (SBCD), and polyvinyl alcohol (PVA). Compared to their parent solid polymers, these flexible supramolecules based on the non-covalent cross-linking of copolymers, SBCD, and PVA efficiently boost the phosphorescence lifetimes (723.0 ms for P-CN, 623.0 ms for P-oM, 945.8 ms for P-mM) through electrostatic interaction and hydrogen bonds. The phosphorescence intensity/lifetime, showing excellent responsiveness to light and heat, sharply decreased after irradiation with a 275 nm flashlight or sunlight and gradually recovered through heating. This is accompanied by the occurrence and fading of visible photochromism, manifesting as dark green for P-CN and pink for P-oM and P-mM. These reversible photochromism and phosphorescence behaviors are mainly attributed to the generation and disappearance of organic radicals in the 4-phenylpyridinium derivatives with a cyano group, which can guide tunable luminescence and photochromism.

Structural biology of microbial gas vesicles: historical milestones and current knowledge.

Gas vesicles mediate buoyancy-based motility in aquatic bacteria and archaea and are the only protein-based structures known to enclose a gas-filled volume. Their unique physicochemical properties and ingenious architecture rank them among the most intriguing macromolecular assemblies characterised to date. This review covers the 60-year journey in quest for a high-resolution structural model of gas vesicles, first highlighting significant strides made in establishing the detailed ultrastructure of gas vesicles through transmission electron microscopy, X-ray fibre diffraction, atomic force microscopy, and NMR spectroscopy. We then survey the recent progress in cryogenic electron microscopy studies of gas vesicles, which eventually led to a comprehensive atomic model of the mature assembly. Synthesising insight from these structures, we examine possible mechanisms of gas vesicle biogenesis and growth, presenting a testable model to guide future experimental work. We conclude by discussing future directions in the structural biology of gas vesicles, particularly considering advancements in AI-driven structure prediction.

Modifying the Catalytic Activity of Lipopeptide Assemblies with Nucleobases.

Biohybrid catalysts that operate in aqueous media are intriguing for systems chemistry. In this paper, we investigate whether control over the self-assembly of biohybrid catalysts can tune their properties. As a model, we use the catalytic activity of functional hybrid molecules consisting of a catalytic H-dPro-Pro-Glu tripeptide, derivatized with fatty acid and nucleobase moieties. This combination of simple biological components merged the catalytic properties of the peptide with the self-assembly of the lipid, and the structural ordering of the nucleobases. The biomolecule hybrids self-assemble in aqueous media into fibrillar assemblies and catalyze the reaction between butanal and nitrostyrene. The interactions between the nucleobases enhanced the order of the supramolecular structures and affected their catalytic activity and stereoselectivity. The results point to the significant control and ordering that nucleobases can provide in the self-assembly of biologically inspired supramolecular catalysts.

Ion-Pairing Assemblies of Anion-Responsive π-Electronic Systems That Have Noncovalently Assisted Expanded Planar Region.

Arylethynyl-substituted dipyrrolyldiketone BF2 complexes as anion-responsive π-electronic molecules exhibited characteristic electronic properties derived from conformation changes upon anion binding, which caused an increase in UV/vis absorption and associated two-photon absorption. The anion complexes showed expanded planar regions assisted by intramolecular interactions, resulting in charge-by-charge ion-pairing assemblies in the solid state.

Polyfluoroalkyl Chain-Based Assemblies for Biomimetic Catalysis.

Amphiphobic fluoroalkyl chains are exploited for creating robust and diverse self-assembled biomimetic catalysts. Long terminal perfluoroalkyl chains (Cn F2n+1 with n=6, 8, and 10) linked with a short perhydroalkyl chains (Cm H2m with m=2 and 3) were used to synthesize several 1,4,7-triazacyclononane (TACN) derivatives, Cn F2n+1 -Cm H2m -TACN. In the presence of an equimolar amount of Zn2+ ions that coordinate the TACN moiety and drive the self-assembly into micelle-like aggregates, the critical aggregation concentration of polyfluorinated Cn F2n+1 -Cm H2m -TACN⋅Zn2+ was lowered by ∼1 order of magnitude compared to the traditional perhyroalkyl counterpart with identical carbon number of alkyl chain. When 2'-hydroxypropyl-4-nitrophenyl phosphate was used as the model phosphate substrate, polyfluorinated Cn F2n+1 -Cm H2m -TACN⋅Zn2+ assemblies showed higher affinity and catalytic activity, compared to its perhyroalkyl chain-based counterpart. Coarse-grained molecular dynamic simulations have been introduced to explore the supramolecular assembly of polyfluoroalkyl chains in the presence of Zn2+ ions and to better understand their enhanced catalytic activity.