nils-herrmann/clean_scirepeval huggingface dataset:
A dynamic view of DNA structure within the nucleosome: biological implications.
Venue: Journal of structural biology

Most of eukaryotic cellular DNA is packed in nucleosome core particles (NCPs), in which the DNA (DNANCP) is wrapped around histones. The influence of this organization on the intrinsic local dynamics of DNA is largely unknown, in particular because capturing such information from experiments remains notoriously challenging. Given the importance of dynamical properties in DNA functions, we addressed this issue using MD simulations of a nucleosome containing the NCP positioning 601 sequence and four related free dodecamers. Comparison between DNANCP and free DNA reveals a limited impact of the dense DNA-histone interface on correlated motions of dinucleotide constituents and on fluctuations of inter base pair parameters. A characteristic feature intimately associated with the DNANCP super-helical path is a set of structural periodicities that includes a marked alternation of regions enriched in backbone BI and BII conformers. This observation led to uncover a convincing correspondence between the sequence effect on BI/BII propensities in both DNANCP and free DNA, strengthening the idea that the histone preference for particular DNA sequences relies on those intrinsic structural properties. These results offer for the first time a detailed view of the DNA dynamical behavior within NCP. They show in particular that the DNANCP dynamics is substantial enough to preserve the ability to structurally adjust to external proteins, for instance remodelers. Also, fresh structural arguments highlight the relevance of relationships between DNA sequence and structural properties for NCP formation. Overall, our work offers a more rational framework to approach the functional, biological roles of NCP.
The shell matrix and microstructure of the Ram's Horn squid: molecular and structural characterization.
Venue: Journal of structural biology

Molluscs are one of the most diversified phyla among metazoans. Most of them produce an external calcified shell, resulting from the secretory activity of a specialized epithelium of the calcifying mantle. This biomineralization process is controlled by a set of extracellular macromolecules, the organic matrix. In spite of several studies, these components are mainly known for bivalves and gastropods. In the present study, we investigated the physical and biochemical properties of the internal planispiral shell of the Ram's Horn squid Spirula spirula. Scanning Electron Microscope investigations of the shell reveal a complex microstructural organization. The saccharides constitute a quantitatively important moiety of the matrix, as shown by Fourier-transform infrared spectroscopy. Solid-state nuclear magnetic resonance spectroscopy identified β-chitin and additional polysaccharides for a total amount of 80% of the insoluble fraction. Proteomics was applied to both soluble and insoluble matrices and in silico searches were performed, first on heterologous metazoans models, and secondly on an unpublished transcriptome of Spirula spirula. In the first case, several peptides were identified, some of them matching with tyrosinase, chitinase 2, protease inhibitor, or immunoglobulin. In the second case, 39 hits were obtained, including transferrin, a serine protease inhibitor, matrilin, or different histones. The very few similarities with known molluscan shell matrix proteins suggest that Spirula spirula uses a unique set of shell matrix proteins for constructing its internal shell. The absence of similarity with closely related cephalopods demonstrates that there is no obvious phylogenetic signal in the cephalopod skeletal matrix.
Microstructural and compositional variation in pacu and piranha teeth related to diet specialization (Teleostei: Serrasalmidae).
Venue: Journal of structural biology

In any vertebrate group, tooth shape is known to fit with a biological function related to diet. However, little is known about the relationships between diet and tooth microstructure and composition in teleost fishes. In this work, we describe the external morphology, internal microstructure and elemental composition of the oral teeth of three representative species of the family Serrasalmidae having different feeding habits (herbivorous vs. omnivorous vs. carnivorous). We used backscattered-electron imaging and low vacuum environmental scanning electron microscope to compare the organization and mineralization of tooth layers as well as energy dispersive X-ray microanalysis and Raman microspectrometry to investigate the elemental composition, Ca/P ratio and mineralogy of the most superficial layers. Oral teeth of each serrasalmid species have the same internal organization based on five distinctive layers (i.e. pulp, dentine, inner enameloid, outer enameloid and cuticle) but the general tooth morphology is different according to diet. Microstructural and compositional variation of the cuticle and iron-enrichment of superficial layers were highlighted between herbivorous and carnivorous species. Iron is more concentrated in teeth of the herbivorous species where it is associated with a thicker cuticle explaining the more intense red-pigmentation of the cutting edges of oral teeth. The iron-enrichment is interpreted as a substitution of Ca by Fe in the hydroxyapatite. These traits are discussed in the light of the evolutionary history of the family. Further considerations and hypotheses about the formation and origin of the mineralized tooth layers and especially the iron-rich superficial layers in teleost fishes are suggested.
The Key Role of E418 Carboxyl Group in the Formation of Nt.BspD6I Nickase Active Site: Structural and Functional Properties of Nt.BspD6I E418A Mutant.
Venue: Journal of structural biology

The mutated nickase Nt.BspD6I E418A has been obtained by site-directed mutagenesis. The purified protein has been crystallized, and its spatial structure has been determined at 2.45 Å resolution. An analysis of the crystal structures of the wild-type and mutated nickase have shown that the elimination of a carboxyl group due to the E418A mutation initiates marked conformational changes in both the N-terminal recognition domain and the C-terminal catalytic domain of nickase and insignificantly affects its linker domain. This is supported by changes in the functional properties of mutated nickase: an increase in the oligomerization capacity in the presence of a substrate, a reduction in the capacity to bind a substrate, and complete loss of catalytic activity.
SpeG polyamine acetyltransferase enzyme from Bacillus thuringiensis forms a dodecameric structure and exhibits high catalytic efficiency.
Venue: Journal of structural biology

Polyamines are important for regulating biofilms and the exopolysaccharide of the biofilm matrix of Bacillus subtilis. Understanding how enzymes can regulate polyamine concentrations is critical for learning more about how these processes occur in diverse bacteria. Here, we describe the structure and function of another member of the spermidine/spermine acetyltransferases (SSAT) found in Bacilli. The SpeG enzyme from B. thuringiensis (BtSpeG) binds polyamines in its allosteric site and adopts a dodecameric oligomeric state similar to other SpeG enzymes from Gram-negative bacteria. Our kinetic results show the catalytic efficiency of BtSpeG was greater than any previously characterized SpeG to date, and in contrast to other SpeG proteins it exhibited very similar kinetic properties toward both spermine and spermidine. Similar to the SpeG enzyme from E. coli, BtSpeG was able to acetylate spermidine on the N1 and N8 positions. The turnover of BtSpeG toward spermine and spermidine was also two to three orders of magnitude greater than any other Bacilli SSAT enzyme that has been previously characterized. SpeG proteins from Bacilli, including B. cereus, B. thuringiensis and B. anthracis share nearly identical sequences and therefore our results likely provide insight into the structure/function relationship across multiple Bacillus species.
MicrographCleaner: a python package for cryo-EM micrograph cleaning using deep learning.
Venue: Journal of structural biology

Cryo-EM Single Particle Analysis workflows require tens of thousands of high-quality particle projections to unveil the three-dimensional structure of macromolecules. Conventional methods for automatic particle picking tend to suffer from high false-positive rates, hampering the reconstruction process. One common cause of this problem is the presence of carbon and different types of high-contrast contaminations. In order to overcome this limitation, we have developed MicrographCleaner, a deep learning package designed to discriminate, in an automated fashion, between regions of micrographs which are suitable for particle picking, and those which are not. MicrographCleaner implements a U-net-like deep learning model trained on a manually curated dataset compiled from over five hundred micrographs. The benchmarking, carried out on approximately one hundred independent micrographs, shows that MicrographCleaner is a very efficient approach for micrograph preprocessing. MicrographCleaner (micrograph_cleaner_em) package is available at PyPI and Anaconda Cloud and also as a Scipion/Xmipp protocol. Source code is available at https://github.com/rsanchezgarc/micrograph_cleaner_em.
Megapinosome: Morphological Description of a Novel Organelle.
Venue: Journal of structural biology

The megapinosome is an endocytic cell organel that we observed in human macrophages with electron microscopy. In a previous work we showed that it is formed by an endocytic event that we called megapinocytosis. The megapinosome is filled with a membrane surrounded trabecular meshwork that is topologically part of the cytosol. In this work we used scanning transmission electron tomography on high pressure frozen and freeze substituted human macrophages in order to unravel the three-dimensional structure of both the megapinosome and the adjacent structures. The megapinosome consists of the trabecular meshwork and the lacunae which are connected with and topologically equivalent to the cytosol. The surrounding lumen is topologically equivalent to the structures of the vesicular pathway. In addition, we show the connections of the trabecular meshwork with the cytosol and the connection of the megapinosomes to a complex tubular and cisternal system covering a large part of the macrophages that we named megapinosome complex. We assume that our methodological approach, based on high pressure freezing from a defined physiological state and three-dimensional imaging, renders the tubular components of the macrophages better visible than the classical two-dimensional imaging of chemically fixed cells used as a "blueprint" for textbook illustrations. The cell biological functions of the megapinosome are largely enigmatic. Probably, megapinosomes assures storage of surface membranes that can be promptly made available when a macrophage needs to change shape to move through a tissue, to uptake extracellular material or dead cells as well as to fight against microbes.
Extracellular alpha/beta-hydrolase from Paenibacillus species shares structural and functional homology to Tobacco Salicylic Acid Binding Protein 2.
Venue: Journal of structural biology

An alpha/ beta hydrolase annotated as a putative salicylate esterase within the genome of a species of Paenibacillus previously identified from differential and selective growth on Kraft lignin was structurally and functionally characterised. Feruloyl esterases are key to the degradation of lignin in several bacterial species and although this activity was investigated, no such activity was observed. The crystal structure of the Paenibacillus esterase, here denoted as PnbE, was determined at 1.32 Å resolution, showing high similarity to Nicotiana tabacum salicylic acid binding protein 2 from the protein database. Structural similarities between these two structures across the core domains and key catalytic residues were observed, with superposition of catalytic residues giving an RMSD of 0.5 Å across equivalent Cα atoms. Conversely, the cap domains of PnbE and Nicotiana tabacum SABP2 showed greater divergence with decreased flexibility in the PnbE cap structure. Activity of PnbE as a putative methyl salicylate esterase was supported with binding studies showing affinity for salicylic acid and functional studies showing methyl salicylate esterase activity. We hypothesise that this activity could enrich Paenibacillus sp. within the rhizosphere by increasing salicylic acid concentrations within the soil.
The shell matrix of the European thorny oyster, Spondylus gaederopus: microstructural and molecular characterization.
Venue: Journal of structural biology

Molluscs, the largest marine phylum, display extraordinary shell diversity and sophisticated biomineral architectures. However, mineral-associated biomolecules involved in biomineralization are still poorly characterized. We report the first comprehensive structural and biomolecular study of Spondylus gaederopus, a pectinoid bivalve with a peculiar shell texture. Used since prehistoric times, this is the best-known shell of Europe's cultural heritage. We find that Spondylus microstructure is very poor in mineral-bound organics, which are mostly intercrystalline and concentrated at the interface between structural layers. Using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) we characterized several shell protein fractions, isolated following different bleaching treatments. Several peptides were identified as well as six shell proteins, which display features and domains typically found in biomineralized tissues, including the prevalence of intrinsically disordered regions. It is very likely that these sequences only partially represent the full proteome of Spondylus, considering the lack of genomics data for this genus and the fact that most of the reconstructed peptides do not match with any known shell proteins, representing consequently lineage-specific sequences. This work sheds light onto the shell matrix involved in the biomineralization in spondylids. Our proteomics data suggest that Spondylus has evolved a shell-forming toolkit, distinct from that of other better studied pectinoids - fine-tuned to produce shell structures with high mechanical properties, while limited in organic content. This study therefore represents an important milestone for future studies on biomineralized skeletons and provides the first reference dataset for forthcoming molecular studies of Spondylus archaeological artifacts.
Correlations within polyprotein forced unfolding dwell-times introduces sequential dependency.
Venue: Journal of structural biology

Polyproteins, comprised from proteins arrayed in tandem, respond to mechanical loads through partial unfolding and extension. This response to tension that enables their physiological function is related to the ability to dynamically regulate their elasticity. The unique arrangement of their individual mechanical components (proteins and polymeric linkers), and the interactions between them eventually determines their performance. The sequential unfolding-times within a polyprotein are inherently assumed to be independent and identically distributed (iid), thus expected to follow an exponential distribution. Nevertheless, a large body of literature using single molecule force spectroscopy (SMFS) provides evidence that forced unfolding-times of N proteins within a polyprotein do not follow the exponential distribution. Here we use SMFS with Atomic Force Microscopy to measure the unfolding kinetics of Poly-(I91)8 at 180 pN. The unfolding time-intervals were statistically analysed using three common approaches, all exhibiting an N-effect: hierarchical behavior with non-identical unfolding time distributions. Using continuous time random walk approach indicates that the unfolding times display subdiffusive features. Put together with free-energy reconstruction of the whole unfolding polyprotein, we provide physical explanation for this nontrivial behavior, according to which the elongating polypeptide chain with each unfolding event intervenes with the sequential unfolding probabilities and correlates them.
Insights into the stability and substrate specificity of the E. coli aerobic β-oxidation trifunctional enzyme complex.
Venue: Journal of structural biology

Degradation of fatty acids by the β-oxidation pathway results in the formation of acetyl-CoA which enters the TCA cycle for the production of ATP. In E. coli the last three steps of the β-oxidation are catalyzed by two heterotetrameric α2β2 enzymes namely the aerobic trifunctional enzyme (EcTFE) and the anaerobic TFE (anEcTFE). The α subunit of TFE has 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities whereas the β subunit is a thiolase with 3-ketoacyl-CoA thiolase (KAT) activity. Recently, it has been shown that the two TFEs have complementary substrate specificities allowing for the complete degradation of long chain fatty acyl-CoAs into acetyl-CoA under aerobic conditions. Also, it has been shown that the tetrameric EcTFE and anEcTFE assemblies are similar to the TFEs of Pseudomans fragi and human, respectively. Here the properties of the EcTFE subunits are further characterized. Strikingly, it is observed that when expressed separately, EcTFE-α is a catalytically active monomer whereas EcTFE-β is inactive. However, when mixed together active EcTFE tetramer is reconstituted. The crystal structure of the EcTFE-α chain is also reported, complexed with ATP, bound in its HAD active site. Structural comparisons show that the EcTFE hydratase active site has a relatively small fatty acyl tail binding pocket when compared to other TFEs in good agreement with its preferred specificity for short chain 2E-enoyl-CoA substrates. Furthermore, it is observed that millimolar concentrations of ATP destabilize the EcTFE complex, and this may have implications for the ATP-mediated regulation of β-oxidation in E. coli.
Structure-based design approach to rational site-directed mutagenesis of β-lactoglobulin.
Venue: Journal of structural biology

Recombinant proteins play an important role in medicine and have diverse applications in industrial biotechnology. Lactoglobulin has shown great potential for use in targeted drug delivery and body fluid detoxification because of its ability to bind a variety of molecules. In order to modify the biophysical properties of β-lactoglobulin, a series of single-site mutations were designed using a structure-based approach. A 3-dimensional structure alignment of homologous molecules led to the design of nine β-lactoglobulin variants with mutations introduced in the binding pocket region. Seven stable and correctly folded variants (L39Y, I56F, L58F, V92F, V92Y, F105L, M107L) were thoroughly characterized by fluorescence, circular dichroism, isothermal titration calorimetry, size-exclusion chromatography, and X-ray structural investigations. The effects of the amino acid substitutions were observed as slight rearrangements of the binding pocket geometry, but they also significantly influenced the global properties of the protein. Most of the mutations increased the thermal/chemical stability without altering the dimerization constant or pH-dependent conformational behavior. The crystal structures reveal that the I56F and F105L mutations reduced the depth of the binding pocket, which is advantageous since it can reduce the affinity to endogenous fatty acids. The F105L mutant created a unique binding mode for a fatty acid, supporting the idea that lactoglobulin can be altered to bind unique molecules. Selected variants possessing a unique combination of their individual properties can be used for further, more advanced mutagenesis, and the presented results support further research using β-lactoglobulin as a therapeutic delivery agent or a blood detoxifying molecule.
ISCU interacts with NFU1, and ISCU[4Fe-4S] transfers its Fe-S cluster to NFU1 leading to the production of holo-NFU1
Venue: Journal of structural biology

NFU1 is a late-acting factor in the biogenesis of human mitochondrial iron-sulfur proteins. Mutations in NFU1 are associated with genetic diseases such as multiple mitochondrial dysfunctions syndrome 1 (MMDS1) that involve defects in mitochondrial [4Fe-4S] proteins. We present results from NMR spectroscopy, small angle X-ray scattering, size exclusion chromatography, and isothermal titration calorimetry showing that the structured conformer of human ISCU binds human NFU1. The dissociation constant determined by ITC is Kd = 1.1 ± 0.2 μM. NMR and SAXS studies led to a structural model for the complex in which the cluster binding region of ISCU interacts with two α-helices in the C-terminal domain of NFU1. In vitro experiments demonstrate that ISCU[4Fe-4S] transfers its Fe-S cluster to apo-NFU1, in the absence of a chaperone, leading to the assembly of holo-NFU1. By contrast, the cluster of ISCU[2Fe-2S] remains bound to ISCU in the presence of apo-NFU1.
Nanostructure of mouse otoconia.
Venue: Journal of structural biology

Mammalian otoconia of the inner ear vestibular apparatus are calcium carbonate-containing mineralized structures critical in maintaining balance and detecting linear acceleration. The mineral phase of otoconia is calcite, which coherently diffracts X-rays much like a single-crystal. Otoconia contain osteopontin (OPN), a mineral-binding protein influencing mineralization processes in bones, teeth and avian eggshells, for example, and in pathologic mineral deposits. Here we describe mineral nanostructure and the distribution of OPN in mouse otoconia. Scanning electron microscopy and atomic force microscopy of intact and cleaved mouse otoconia revealed an internal nanostructure (∼50 nm). Transmission electron microscopy and electron tomography of focused ion beam-prepared sections of otoconia confirmed this mineral nanostructure, and identified even smaller (∼10 nm) nanograin dimensions. X-ray diffraction of mature otoconia (8-day-old mice) showed crystallite size in a similar range (73 nm and smaller). Raman and X-ray absorption spectroscopy - both methods being sensitive to the detection of crystalline and amorphous forms in the sample - showed no evidence of amorphous calcium carbonate in these mature otoconia. Scanning and transmission electron microscopy combined with colloidal-gold immunolabeling for OPN revealed that this protein was located at the surface of the otoconia, correlating with a site where surface nanostructure was observed. OPN addition to calcite growing in vitro produced similar surface nanostructure. Finally, these findings provide details on the composition and nanostructure of mammalian otoconia, and suggest that while OPN may influence surface rounding and surface nanostructure in otoconia, other incorporated proteins (also possibly including OPN) likely participate in creating internal nanostructure.