nils-herrmann/clean_scirepeval huggingface dataset:
Structural insights into enzymatic activity and substrate specificity determination by a single amino acid in nitrilase from Syechocystis sp. PCC6803.
Venue: Journal of structural biology

Nitrilases are enzymes widely expressed in prokaryotes and eukaryotes that utilize a Cys–Glu–Lys catalytic triad to hydrolyze non-peptide carbon–nitrogen bonds. Nitrilase from Syechocystis sp. Strain PCC6803 (Nit6803) shows hydrolysis activity towards a broad substrate spectrum, ranging from mononitriles to dinitriles and from aromatic nitriles to aliphatic nitriles. Yet, the structural principle of the substrate specificity of this nitrilase is still unknown. We report the crystal structure of Nit6803 at 3.1 Å resolution and propose a structural mechanism of substrate selection. Our mutagenesis data exhibited that the aromaticity of the amino acid at position 146 of Nit6803 is absolutely required for its nitrilase activity towards any substrates tested. Moreover, molecular docking and dynamic simulation analysis indicated that the distance between the sulfhydryl group of the catalytic cysteine residue and the cyano carbon of the substrate plays a crucial role in determining the nitrilase catalytic activity of Nit6803 and its mutants towards different nitrile substrates.
Particle migration analysis in iterative classification of cryo-EM single-particle data.
Venue: Journal of structural biology

Recently developed classification methods have enabled resolving multiple biological structures from cryo-EM data collected on heterogeneous biological samples. However, there remains the problem of how to base the decisions in the classification on the statistics of the cryo-EM data, to reduce the subjectivity in the process. Here, we propose a quantitative analysis to determine the iteration of convergence and the number of distinguishable classes, based on the statistics of the single particles in an iterative classification scheme. We start the classification with more number of classes than anticipated based on prior knowledge, and then combine the classes that yield similar reconstructions. The classes yielding similar reconstructions can be identified from the migrating particles (jumpers) during consecutive iterations after the iteration of convergence. We therefore termed the method "jumper analysis", and applied it to the output of RELION 3D classification of a benchmark experimental dataset. This work is a step forward toward fully automated single-particle reconstruction and classification of cryo-EM data.
Dark-field transmission electron microscopy of cortical bone reveals details of extrafibrillar crystals.
Venue: Journal of structural biology

In a previous study we showed that most of the mineral in bone is present in the form of "mineral structures", 5-6nm-thick, elongated plates which surround and are oriented parallel to collagen fibrils. Using dark-field transmission electron microscopy, we viewed mineral structures in ion-milled sections of cortical human bone cut parallel to the collagen fibrils. Within the mineral structures we observe single crystals of apatite averaging 5.8±2.7nm in width and 28±19nm in length, their long axes oriented parallel to the fibril axis. Some appear to be composite, co-aligned crystals as thin as 2nm. From their similarity to TEM images of crystals liberated from deproteinated bone we infer that we are viewing sections through platy crystals of apatite that are assembled together to form the mineral structures.
Crystal structure of Legionella pneumophila dephospho-CoA kinase reveals a non-canonical conformation of P-loop.
Venue: Journal of structural biology

Dephospho-CoA kinase (DPCK; EC 2.7.1.24) catalyzes the final step in the coenzyme A biosynthetic pathway. DPCK transfers a phosphate group from ATP to the 3-hydroxyl group of the ribose of dephosphocoenzyme A (dCoA) to yield CoA and ADP. Upon the binding of ligands, large conformational changes is induced in DPCKs, as well as in many other kinases, to shield the bound ATP in their catalytic site from the futile hydrolysis by bulk water molecules. To investigate the molecular mechanisms underlying the phosphoryl transfer during DPCK catalytic cycle, we determined the crystal structures of the Legionellapneumophila DPCK (LpDPCK) both in its apo-form and in complex with ATP. The structures reveal that LpDPCK comprises of three domains, the classical core domain, the CoA domain, and the LID domain, which are packed together to create a central cavity for substrate-binding and enzymatic catalysis. The binding of ATP induces large conformational changes, including a hinge-bending motion of the CoA binding domain and the "helix to loop" conformational change of the P-loop. Finally, modeling of a dCoA molecule to the enzyme provides insights into the catalytic mechanism of DPCK.
The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution.
Venue: Journal of structural biology

Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in controlling growth and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, there is limited information on the quaternary structure of LRAP. Here, sedimentation velocity analytical ultracentrifugation (SV) and small angle neutron scattering (SANS) were used to study the tertiary and quaternary structure of LRAP at various pH values, ionic strengths, and concentrations. We found that the monomer is the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7-4.1, pH 4.5-8, 50 mmol/L(mM) to 200 mM NaCl, 0.065-2 mg/mL). The monomer is also the dominant species for unphosphorylated LRAP (LRAP(-P)) at pH 7.4 and for LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregates in a narrow pH range near the isoelectric point of pH 4.1. SV and SANS show that the LRAP monomer has a radius of ∼2.0 nm and an asymmetric structure, and solution NMR studies indicate that the monomer is largely unstructured. This work provides new insights into the secondary, tertiary, and quaternary structure of LRAP in solution and provides evidence that the monomeric species may be an important functional form of some amelogenins.
Thalidomide mimics uridine binding to an aromatic cage in cereblon.
Venue: Journal of structural biology

Thalidomide and its derivatives lenalidomide and pomalidomide are important anticancer agents but can cause severe birth defects via an interaction with the protein cereblon. The ligand-binding domain of cereblon is found, with a high degree of conservation, in both bacteria and eukaryotes. Using a bacterial model system, we reveal the structural determinants of cereblon substrate recognition, based on a series of high-resolution crystal structures. For the first time, we identify a cellular ligand that is universally present: we show that thalidomide and its derivatives mimic and compete for the binding of uridine, and validate these findings in vivo. The nature of the binding pocket, an aromatic cage of three tryptophan residues, further suggests a role in the recognition of cationic ligands. Our results allow for general evaluation of pharmaceuticals for potential cereblon-dependent teratogenicity.
Amino acid sequence homologies in the hard keratins of birds and reptiles, and their implications for molecular structure and physical properties.
Venue: Journal of structural biology

Avian and reptilian epidermal appendages such as feathers, claws and scales exhibit a filament-matrix texture. Previous studies have established that both components reside within the same single-chain molecule. In the present study the homology in a wide range of aligned sequences is used to gain insights into the structure and function of the molecular segments associated with the filament and with the matrix. The notion that all molecules contain a β-rich 34-residue segment associated with the framework of the filament is reinforced by the present study. In addition, the residues involved in the polymerization of the molecules to form filaments are identified. In the Archosaurs (birds, crocodiles and turtles), and the Squamates (snakes and lizards) segments rich in glycine and tyrosine can be identified in the C-terminal domain. In Rhynocephalians (tuataras) and Squamates a similar segment is inserted at a specific point in the N-terminal domain. In some Archosaurian appendages (both avian and reptilian) segments rich in charged residues and cysteine are found in the N-terminal domain. The likely effect of these segments will be to soften the tissue without compromising its insolubility. The structure and role of the various molecular segments identified in this study and the way in which they might manifest themselves in terms of the physical properties of the particular epidermal appendage in which they appear are also discussed.
Biogenic nanospheres of amorphous carbonated Ca-Mg phosphate within the periostracum of the green mussel Perna viridis.
Venue: Journal of structural biology

Recently there is increasing evidence that the shell biomineralization proceeds via an amorphous precursor route. Therefore, the search for and investigation of amorphous biominerals in bivalve shells are of great importance and interest. Here, using a scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffractometer (XRD), and Fourier transform infrared spectrometer (FTIR), we investigate the microstructure and mineralogy of the periostracum in Perna viridis. We find that: (1) the periostracum has three layers, of which the inner and outer layer are of proteins, while the middle layer is mineralized with nanospheres of amorphous biominerals; (2) the nanospheres are of amorphous carbonated Ca-Mg phosphate (ACCP), where the CO3(2)(-)/PO4(3)(-) weight ratio is estimated to be ∼0.3, and the Ca/P and Ca/Mg atomic ratio is ∼1.4 and 1.6, respectively; (3) the nanospheres, with a diameter of 43-106nm, are found to assemble into spherules with a diameter of 160-500nm, which are further organized into parallel microlayers separated by the proteins; and (4) the nanospheres are assumed to function as the pH stabilizer to facilitate the shell's initial mineralization. Finally, we expect that these findings will advance our understanding of the shell's biomineralization process.
Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.
Venue: Journal of structural biology

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.
Quantitative analysis of mouse pancreatic islet architecture by serial block-face SEM.
Venue: Journal of structural biology

We have applied serial block-face scanning electron microscopy (SBF-SEM) to measure parameters that describe the architecture of pancreatic islets of Langerhans, microscopic endocrine organs that secrete insulin and glucagon for control of blood glucose. By analyzing entire mouse islets, we show that it is possible to determine (1) the distributions of alpha and beta cells, (2) the organization of blood vessels and pericapillary spaces, and (3) the ultrastructure of the individual secretory cells. Our results show that the average volume of a beta cell is nearly twice that of an alpha cell, and the total mitochondrial volume is about four times larger. In contrast, nuclear volumes in the two cell types are found to be approximately equal. Although the cores of alpha and beta secretory granules have similar diameters, the beta granules have prominent halos resulting in overall diameters that are twice those of alpha granules. Visualization of the blood vessels revealed that every secretory cell in the islet is in contact with the pericapillary space, with an average contact area of 9±5% of the cell surface area. Our data show that consistent results can be obtained by analyzing small numbers of islets. Due to the complicated architecture of pancreatic islets, such precision cannot easily be achieved by using TEM of thin sections.
Structure of the c-Src-SH3 domain in complex with a proline-rich motif of NS5A protein from the hepatitis C virus.
Venue: Journal of structural biology

The non-structural hepatitis C virus proteins NS5A and NS5B form a complex through interaction with the SH2 and SH3 domains of the non-receptor Src tyrosine kinase, which seems essential for viral replication. We have crystallized the complex between the SH3 domain of the c-Src tyrosine kinase and the C-terminal proline rich motif of the NS5A protein (A349PPIPPPRRKR359). Crystals obtained at neutral pH belong to the space group I41, with a single molecule of the SH3/NS5A complex at the asymmetric unit. The NS5A peptide is bound in a reverse orientation (class II) and the comparison of this structure with those of the high affinity synthetic peptides APP12 and VSL12 shows some important differences at the salt bridge that drives the peptide orientation. Further conformational changes in residues placed apart from the binding site also seem to play an important role in the binding orientation of this peptide. Our results show the interaction of the SH3 domain of the c-Src tyrosine kinase with a proline rich motif in the NS5A protein and point to their potential interaction in vivo.
Layered growth of crayfish gastrolith: about the stability of amorphous calcium carbonate and role of additives.
Venue: Journal of structural biology

Previous studies on pre-molt gastroliths have shown a typical onion-like morphology of layers of amorphous mineral (mostly calcium carbonate) and chitin, resulting from the continuous deposition and densification of amorphous mineral spheres on a chitin-matrix during time. To investigate the consequences of this layered growth on the local structure and composition of the gastrolith, we performed spatially-resolved Raman, X-ray and SEM-EDS analysis on complete pre-molt gastrolith cross-sections. Results show that especially the abundance of inorganic phosphate, phosphoenolpyruvate (PEP)/citrate and proteins is not uniform throughout the organ but changes from layer to layer. Based on these results we can conclude that ACC stabilization in the gastrolith takes place by more than one compound and not by only one of these additives.
The response of the pediatric head to impacts onto a rigid surface.
Venue: Journal of biomechanics

The study of pediatric head injury relies heavily on the use of finite element models and child anthropomorphic test devices (ATDs). However, these tools, in the context of pediatric head injury, have yet to be validated due to a paucity of pediatric head response data. The goal of this study is to investigate the response and injury tolerance of the pediatric head to impact. Twelve pediatric heads were impacted in a series of drop tests. The heads were dropped onto five impact locations (forehead, occiput, vertex and right and left parietal) from drop heights of 15 and 30 cm. The head could freely fall without rotation onto a flat 19 mm thick platen. The impact force was measured using a 3-axis piezoelectric load cell attached to the platen. Age and drop height were found to be significant factors in the impact response of the pediatric head. The head acceleration (14-15 cm; 103-30 cm), Head Injury Criterion (HIC) (253-15 cm; 154-30 cm) and impact stiffness (5800-15 cm; 3755-30 cm) when averaged across all impact locations increased with age from 33 weeks gestation to 16 years, while the pulse duration (66-15 cm; 53-30 cm) decreased with age. Increases in head acceleration, HIC and impact stiffness were also observed with increased drop height, while pulse duration decreased with increased drop height. One important observation was that three of the four cadaveric heads between the ages of 5-months and 22-months sustained fractures from the 15 cm and 30 cm drop heights. The 5-month-old sustained a right parietal linear fracture while the 11- and 22-month-old sustained diastatic linear fractures.
Depletion of glycosphingolipids induces excessive response of chondrocytes under mechanical stress.
Venue: Journal of biomechanics

Glycosphingolipids (GSLs) are ubiquitous membrane components that play an indispensable role in maintaining chondrocyte homeostasis. To gain better insight into roles of GSLs, we studied the effects of GSL-deletion on the physiological responses of chondrocytes to mechanical stress. Mice lacking Ugcg gene (Ugcg-/-) were genetically generated to obtain GSL-deficient mice, and their chondrocytes from the joints were used for functional analyses in vitro culture experiments. The cells were seeded in a three-dimensional collagen gel and subjected to 5%, 10% or 16% cyclic tensile strain for either 3 or 24 h. The gene expressions of chondrocyte anabolic and catabolic factors, and the induction of Ca2+ signaling were analyzed. Our results revealed that chondrocytes derived from GSL-deficient mice exhibited an elevation in the expression of catabolic factors (ADAMTS-5, MMP-13) following the exposure to strain with amplitudes of 10%. Likewise, applying cyclic tensile strain with these amplitudes resulted in an increased Ca2+ oscillation ratio in chondrocytes from GSL-deficient as compared to the ratio from control mice. These results demonstrated that deletion of GSL stimulated the catabolic responses of chondrocytes to mechanical stress via the augmentation of the sensitivity to mechanical stress that may lead to the cartilage deterioration. These findings suggest that the regulation of the physiological responses of chondrocytes by GSLs could be a potential target in a therapeutic intervention in osteoarthritis.
Biaxial biomechanical properties of the nonpregnant murine cervix and uterus.
Venue: Journal of biomechanics

From a biomechanical perspective, female reproductive health is an understudied area of research. There is an incomplete understanding of the complex function and interaction between the cervix and uterus. This, in part, is due to the limited research into multiaxial biomechanical functions and geometry of these organs. Knowledge of the biomechanical function and interaction between these organs may elucidate etiologies of conditions such as preterm birth. Therefore, the objective of this study was to quantify the multiaxial biomechanical properties of the murine cervix and uterus using a biaxial testing set-up. To accomplish this, an inflation-extension testing protocol (n = 15) was leveraged to quantify biaxial biomechanical properties while preserving native matrix interactions and geometry. Ultrasound imaging and histology (n = 10) were performed to evaluate regional geometry and microstructure, respectively. Histological analysis identified a statistically significant greater collagen content and significantly smaller smooth muscle content in the cervix as compared to the uterus. No statistically significant differences in elastic fibers were identified. Analysis of bilinear fits revealed a significantly stiffer response from the circumferentially orientated ECM fibers compared to axially orientated fibers in both organs. Bilinear fits and a two-fiber family constitutive model showed that the cervix was significantly less distensible than the uterus. We submit that the regional biaxial information reported in this study aids in establishing an appropriate reference configuration for mathematical models of the uterine-cervical complex. Thus, may aid future work to elucidate the biomechanical mechanisms leading to cervical or uterine conditions.
Vibration settling time of the gastrocnemius remains constant during an exhaustive run in rear foot strike runners.
Venue: Journal of biomechanics

In this study, vibrations of human gastrocnemius during an exhaustive run protocol are considered for analysis. Previous studies have shown increased vibration intensity and damping coefficient within the soft tissue with fatigue. The question of this study was to investigate if the vibration settling time remains constant during a prolonged running. Eleven semi-professional middle/long distance runners ran to exhaustion on a treadmill with their preferred constant speed (4.29 ± 0.33 m/s) for 3873 ± 1147 m. Vibration of the gastrocnemius lateralis, electrical activity of the tibialis anterior and the gastrocnemius medialis along with ground reaction force (GRF) were recorded. The results demonstrated significant increase in impact peak and loading rate, and the frequency content of the impact, with no significant change in active peak of the vertical GRF. Fatigue resulted in increased vibration intensity, damping coefficient, and energy dissipation of vibration with no change in vibration settling time. Furthermore, peak acceleration significantly linearly (R = 0.59) increased as a function of running time. The mean frequency of muscle activity of the gastrocnemius medialis and the intensity of muscle activity in TA significantly decreased. The results suggest that constant vibration settling time might either be an objective for muscle tuning which is more pronounced in fatigued state or a passive by-product of muscle function in running. Further studies are needed to address this point.