Improving fragment-based ab initio protein structure assembly using low-accuracy contact-map predictions

Sequence-based contact prediction has shown considerable promise in assisting non-homologous structure modeling, but it often requires many homologous sequences and a sufficient number of correct contacts to achieve correct folds. Here, we developed a method, C-QUARK, that integrates multiple deep-learning and coevolution-based contact-maps to guide the replica-exchange Monte Carlo fragment assembly simulations. The method was tested on 247 non-redundant proteins, where C-QUARK could fold 75% of the cases with TM-scores (template-modeling scores) ≥0.5, which was 2.6 times more than that achieved by QUARK. For the 59 cases that had either low contact accuracy or few homologous sequences, C-QUARK correctly folded 6 times more proteins than other contact-based folding methods. C-QUARK was also tested on 64 free-modeling targets from the 13th CASP (critical assessment of protein structure prediction) experiment and had an average GDT_TS (global distance test) score that was 5% higher than the best CASP predictors. These data demonstrate, in a robust manner, the progress in modeling non-homologous protein structures using low-accuracy and sparse contact-map predictions.

Systemic Expression, Purification, and Initial Structural Characterization of Bacteriophage T4 Proteins Without Known Structure Homologs

Bacteriophage T4 of Escherichia coli is one of the most studied phages. Research into it has led to numerous contributions to phage biology and biochemistry. Coding about 300 gene products, this double-stranded DNA virus is the best-understood model in phage study and modern genomics and proteomics. Ranging from viral RNA polymerase, commonly found in phages, to thymidylate synthase, whose mRNA requires eukaryotic-like self-splicing, its gene products provide a pool of fine examples for phage research. However, there are still up to 130 gene products that remain poorly characterized despite being one of the most-studied model phages. With the recent advancement of cryo-electron microscopy, we have a glimpse of the virion and the structural proteins that present in the final assembly. Unfortunately, proteins participating in other stages of phage development are absent. Here, we report our systemic analysis on 22 of these structurally uncharacterized proteins, of which none has a known homologous structure due to the low sequence homology to published structures and does not belong to the category of viral structural protein. Using NMR spectroscopy and cryo-EM, we provided a set of preliminary structural information for some of these proteins including NMR backbone assignment for Cef. Our findings pave the way for structural determination for the phage proteins, whose sequences are mainly conserved among phages. While this work provides the foundation for structural determinations of proteins like Gp57B, Cef, Y04L, and Mrh, other in vitro studies would also benefit from the high yield expression of these proteins.

Structure Unveils Relationships between RNA Virus Polymerases

RNA viruses are the fastest evolving known biological entities. Consequently, the sequence similarity between homologous viral proteins disappears quickly, limiting the usability of traditional sequence-based phylogenetic methods in the reconstruction of relationships and evolutionary history among RNA viruses. Protein structures, however, typically evolve more slowly than sequences, and structural similarity can still be evident, when no sequence similarity can be detected. Here, we used an automated structural comparison method, homologous structure finder, for comprehensive comparisons of viral RNA-dependent RNA polymerases (RdRps). We identified a common structural core of 231 residues for all the structurally characterized viral RdRps, covering segmented and non-segmented negative-sense, positive-sense, and double-stranded RNA viruses infecting both prokaryotic and eukaryotic hosts. The grouping and branching of the viral RdRps in the structure-based phylogenetic tree follow their functional differentiation. The RdRps using protein primer, RNA primer, or self-priming mechanisms have evolved independently of each other, and the RdRps cluster into two large branches based on the used transcription mechanism. The structure-based distance tree presented here follows the recently established RdRp-based RNA virus classification at genus, subfamily, family, order, class and subphylum ranks. However, the topology of our phylogenetic tree suggests an alternative phylum level organization.

Playing Ludomotor Activities in Lleida During the Spanish Civil War: An Ethnomotor Approach

The traditional ludomotor activities (LA) are recognized by UNESCO as an intangible piece of cultural heritage. The ethnomotricity analyzes LA in its sociocultural context, taking into account the proprieties of rules or motor conditions (internal logic) and the link with local culture (external logic). The aim of this research was to identify and reveal the distinctive ethnomotor features of LA in order to understand the adaptations that occurred in the social scenario of the Spanish Civil War (1936–1939) in Lleida. The corpus of the research was constituted by 101 LA which were collected from the analysis of 20 semi-structured interviews. An “ad hoc” tool was designed and agreed upon by expert observers. It was comprised of a total of 27 ethnomotor variables related to LA. The experts achieved high reliability [Cohen’s kappa coefficient (κ) and Spearman’s correlation coefficient = 1] when the classification of LA was carried out on two different occasions. Descriptive statistics, cross-tabulations (Pearson’s chi-squared) effect sizes, and two-step clusters were performed by external and internal logic variables. The presence or absence of motor interaction (X2 = 9.029; df = 1; p < 0.003; ES = 0.298) was enlightening when comparing LA with and without a war connotation. On the other hand, the hierarchy of variables rested primarily on IL-Domain (Psycho-Coop-Oppo-Coop/Oppo) (PI = 1). Among other singularities, while two-step cluster analysis revealed a corresponding ethnomotor silhouette with cluster 1, with the warlike connotation (n = 48; 96.0%), its homologous structure was expressed (Cluster 2) in the absence of the warlike character (n = 26; 50%).

LAHMA: structure analysis through local annotation of homology-matched amino acids

Comparison of homologous structure models is a key step in analyzing protein structure. With a wealth of homologous structures, comparison becomes a tedious process, and often only a small (user-biased) selection of data is used. A multitude of structural superposition algorithms are then typically used to visualize the structures together in 3D and to compare them. Here, the Local Annotation of Homology-Matched Amino acids (LAHMA) website (https://lahma.pdb-redo.eu) is presented, which compares any structure model with all of its close homologs from the PDB-REDO databank. LAHMA displays structural features in sequence space, allowing users to uncover differences between homologous structure models that can be analyzed for their relevance to chemistry or biology. LAHMA visualizes numerous structural features, also allowing one-click comparison of structure-quality plots (for example the Ramachandran plot) and `in-browser' structural visualization of 3D models.

Experimental phasing with vanadium and application to nucleotide-binding membrane proteins

The structure determination of soluble and membrane proteins can be hindered by the crystallographic phase problem, especially in the absence of a suitable homologous structure. Experimental phasing is the method of choice for novel structures; however, it often requires heavy-atom derivatization, which can be difficult and time-consuming. Here, a novel and rapid method to obtain experimental phases for protein structure determination by vanadium phasing is reported. Vanadate is a transition-state mimic of phosphoryl-transfer reactions and it has the advantage of binding specifically to the active site of numerous enzymes catalyzing this reaction. The applicability of vanadium phasing has been validated by determining the structures of three different protein–vanadium complexes, two of which are integral membrane proteins: the rabbit sarcoplasmic reticulum Ca2+-ATPase, the antibacterial peptide ATP-binding cassette transporter McjD from Escherichia coli and the soluble enzyme RNAse A from Bos taurus. Vanadium phasing was successful even at low resolution and despite severe anisotropy in the data. This method is principally applicable to a large number of proteins, representing six of the seven Enzyme Commission classes. It relies exclusively on the specific chemistry of the protein and it does not require any modifications, making it a very powerful addition to the phasing toolkit. In addition to the phasing power of this technique, the protein–vanadium complexes also provide detailed insights into the reaction mechanisms of the studied proteins.

Serial Section-Based 3D Reconstruction of Anaxagorea (Annonaceae) Carpel Vasculature and Implications on Integrated Axial-Foliar Origin of Angiosperm Carpels

The carpel is the basic unit of the gynoecium in angiosperms and one of the most important morphological features distinguishing angiosperms from gymnosperms; therefore, carpel origin is of great significance in angiosperm phylogenetic origin. Recent consensus favors the interpretation that the carpel originates from the fusion of an ovule-bearing axis and the phyllome that subtends it. It has been confirmed by morphological and molecular evidence that foliar homologs are involved in carpel evolution. Consequently, if axial homologs can be traced in the carpel, it would more likely be derived from an integrated axial-foliar structure. This study aimed to reveal the axial structures in carpels by analyzing the continuous changes in vasculature from the receptacle to the carpels and ovules. Anaxagorea is the most basal genus in a primitive angiosperm family, Annonaceae. The conspicuous carpel stipe at the base of each carpel makes it an ideal material for exploring the possible axial homologous structure in the carpel. In this study, floral organogenesis and the topological vasculature structure were delineated in Anaxagorea luzonensis and Anaxagorea javanica, and a 3D-model of the carpel vasculature was reconstructed based on the serial sections. The results show that (1) at the flowering stage, the number of vascular bundles entering each Anaxagorea carpel from the receptacle was significantly higher than three, arranged in a radiosymmetric pattern, and forming a basal ring at the base of each carpel. (2) All carpel bundles were only connected with the central stele. (3) At the slightly upper part of the carpel, all lateral bundles from the basal ring were reorganized into two groups, each forming a lateral bundle complex below the respective placenta. Bundles in each lateral bundle complex were also ringed. (4) The ovule bundles were composed of non-adjacent bundles in the lateral bundle complex. The results of the present study suggest that the circular arrangement of bundles in the receptacle, carpel stipe, and placenta of Anaxagorea are in line with the composite axial-foliar nature of the carpel, and provide a morphological basis for further research on the origin of the carpel.

The Seimei telescope project and technical developments

ABSTRACT An overview of the Seimei telescope, a 3.8 m optical infrared telescope located on Mt. Chikurinji in the Okayama prefecture of Japan, is presented. Seimei is a segmented-mirror telescope whose primary mirror consists of 18 petal-shaped segments. The telescope tube supporting the thin segmented mirrors is structurally incorporated within large arc-rails providing the elevation axis. The tube has a light-weight homologous structure designed with a genetic algorithm. The total weight of the telescope tube, including 1.4-ton optics, is only 8 tons. By virtue of its light weight, the telescope is able to point at an object anywhere in the observable sky within one minute. The telescope is operated by Kyoto University in collaboration with the National Astronomical Observatory of Japan (NAOJ). Half of the telescope time is used by Kyoto University. The remaining time is open to the Japanese astronomical community. NAOJ is responsible for the management of the open-use time, including handling of the observation proposals. The telescope is now regularly performing scientific observations on the basis of a variety of proposals.

Comparative Contemplations on the Hippocampus

The hippocampus in mammals is a morphologically well-defined structure, and so are its main subdivisions. To define the homologous structure in other vertebrate clades, using these morphological criteria has been difficult, if not impossible, since the typical mammalian morphology is absent. Although there seems to be consensus that the most medial part of the pallium represents the hippocampus in all vertebrates, there is no consensus on whether all mammalian hippocampal subdivisions are present in the derivatives of the medial pallium in all vertebrate groups. The aim of this paper is to explore the potential relevance of connections to define the hippocampus across vertebrates, with a focus on mammals, reptiles, and birds.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

ABSTRACT Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. IMPORTANCE To fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available.