Additional Restraints Research Articles

NMR “Crystallography” for Uniformly (13C, 15N)‐Labeled Oriented Membrane Proteins

In oriented-sample (OS) solid-state NMR of membrane proteins, the angular-dependent dipolar couplings and chemical shifts provide a direct input for structure calculations. However, so far only 1 H-15 N dipolar couplings and 15 N chemical shifts have been routinely assessed in oriented 15 N-labeled samples. The main obstacle for extending this technique to membrane proteins of arbitrary topology has remained in the lack of additional experimental restraints. We have developed a new experimental triple-resonance NMR technique, which was applied to uniformly doubly (15 N, 13 C)-labeled Pf1 coat protein in magnetically aligned DMPC/DHPC bicelles. The previously inaccessible 1 Hα -13 Cα dipolar couplings have been measured, which make it possible to determine the torsion angles between the peptide planes without assuming α-helical structure a priori. The fitting of three angular restraints per peptide plane and filtering by Rosetta scoring functions has yielded a consensus α-helical transmembrane structure for Pf1 protein.

A Molecular Mechanics Model for Flavins.

Flavin containing molecules form a group of important cofactors that assist a wide range of enzymatic reactions. Flavins use the redox-active isoalloxazine system, which is capable of one- and two-electron transfer reactions and can exist in several protonation states. In this work, molecular mechanics force field parameters compatible with the CHARMM36 all-atom additive force field were derived for biologically important flavins, including riboflavin, flavin mononucleotide, and flavin adenine dinucleotide. The model was developed for important protonation and redox states of the isoalloxazine group. The partial charges were derived using the CHARMM force field parametrization strategy, where quantum mechanics water-solute interactions are used to target optimization. In addition to monohydrate energies and geometries, electrostatic potential around the compound was used to provide additional restraints during the charge optimization. Taking into account the importance of flavin-containing molecules special attention was given to the quality of bonded terms. All bonded terms, including stiff terms and torsion angle parameters, were parametrized using exhaustive potential energy surface scans. In particular, the model reproduces well the butterfly motion of isoalloxazine in the oxidized and reduced forms as predicted by quantum mechanics in gas phase. The model quality is illustrated by simulations of four flavoproteins. Overall, the presented molecular mechanics model will be of utility to model flavin cofactors in different redox states. © 2019 Wiley Periodicals, Inc.

ASSESSMENT OF THE EFFICIENCY OF THE USE OF ADDITIONAL HOLDING DEVICES TO IMPROVE THE STABILITY OF SHEET PILES MADE OF POLYVINYLCHLORIDE

Objectives. This article discusses options for additional restraints for support structures made from polymer dowels. The issue of increasing the stability of polymer sheet piles plays a key role in the design and construction of facilities using this type of material. The article compares two variants of the restraining devices and compares them with the non-reinforced version using the example of a notch of the same depth.Methods. The calculations are carried out by the Coulomb- Mohr methods implemented in the GEO5 software package module “Pit Fencing - Analysis.Result. The obtained calculations showed the possibility of using both types of restraints in retaining structures made of polymer dowels. Conclusion. Calculations have shown the effectiveness of both methods in their use as additional restraints for supporting structures using PVC dowels.The variant with the installation of the support pipe allows to slightly reduce the deformation of the sheet pile structure, but it does not have the ability to adjust the vertical position of the wall, which is present in the variant with the anchor system by welding the threaded rods to the ends of the metal braces or using talreeps.

QRNAS: software tool for refinement of nucleic acid structures

BackgroundComputational models of RNA 3D structure often present various inaccuracies caused by simplifications used in structure prediction methods, such as template-based modeling or coarse-grained simulations. To obtain a high-quality model, the preliminary RNA structural model needs to be refined, taking into account atomic interactions. The goal of the refinement is not only to improve the local quality of the model but to bring it globally closer to the true structure.ResultsWe present QRNAS, a software tool for fine-grained refinement of nucleic acid structures, which is an extension of the AMBER simulation method with additional restraints. QRNAS is capable of handling RNA, DNA, chimeras, and hybrids thereof, and enables modeling of nucleic acids containing modified residues.ConclusionsWe demonstrate the ability of QRNAS to improve the quality of models generated with different methods. QRNAS was able to improve MolProbity scores of NMR structures, as well as of computational models generated in the course of the RNA-Puzzles experiment. The overall geometry improvement may be associated with increased model accuracy, especially on the level of correctly modeled base-pairs, but the systematic improvement of root mean square deviation to the reference structure should not be expected. The method has been integrated into a computational modeling workflow, enabling improved RNA 3D structure prediction.

Glutton: a tool for generating structural ensembles of partly disordered proteins from chemical shifts.

Many proteins are partially disordered in physiological conditions and only fold, fully or partially, upon binding. Their structural analysis is challenging because the accessible information, typically chemical shifts (CS) from nuclear magnetic resonance experiments, are averages over broad ensembles of conformations. We aim to develop a database for the analysis of such data in terms of conformational distributions of the protein backbone rather than of individual high-resolution structures. Glutton is the largest available database linking CS and protein 3D structures (5270 entries organized in three levels) and is searchable via a python script. It generates statistical distributions of ϕ-ψ dihedral angles based on CS or vice versa. Such ϕ-ψ distributions are used to calculate structural ensembles of partially disordered proteins from their CS. For folded proteins, such ensembles are excellent starting points for further refinement with additional experimental restraints (structure determination) or computational methods (structure prediction). Glutton is freely available at https://github.com/YeeHo/Glutton. Supplementary data are available at Bioinformatics online.

Numerical Analysis and 1D/2D Sensitivity Study for Monolithic and Laminated Structural Glass Elements under Thermal Exposure.

Glass is largely used in architectural and engineering applications (i.e., buildings and vehicles) as a structural material, especially in the form of laminated glass (LG) sections. To achieve adequate and controlled safety levels in these applications, the well-known temperature-dependent behavior of viscoelastic interlayers for LG sections should be properly accounted for during the design process. Furthermore, the materials’ thermomechanical degradation with increases of temperature could severely affect the load-bearing performance of glass assemblies. In this context, uncoupled thermomechanical finite element (FE) numerical models could represent a robust tool and support for design engineers. Key input parameters and possible limits of the FE method, however, should be properly calibrated and assessed, so as to enable reliable estimations for the real behavior of glazing systems. In this paper, FE simulations are proposed for monolithic (MG) and LG specimens under radiant heating, based on one-dimensional (1D) and two-dimensional (2D) models. A special attention is focused on thermal effects, being representative of the first step for conventional uncoupled, thermomechanical analyses. Based on experimental results available in the literature, FE parametric studies are discussed, giving evidence of limits and issues due to several modeling assumptions. In particular, careful consideration is paid for various thermal material properties (conductivity, specific heat) and thermal boundaries (conductivity, emissivity), but also for other influencing parameters like the geometrical features of samples (thickness tolerances, cross-sectional properties, etc.), the composition of LG sections (interlayer type, thickness), the loading pattern (heat transfer distribution) and the presence of additional mechanical restraints (i.e., supports of different materials). Comparative FE results are hence critically discussed, highlighting the major effects of such influencing parameters.

Three-Dimensional Composite Floor Systems under Column-Removal Scenarios

In this study, four 1/3-scaled enhanced steel frame–composite floor specimens were tested quasi-statically under a column-removal scenario. One specimen was the control test and the other three were different in terms of aspect ratio, degree of composite action, and boundary condition. The objective of this paper is to study the effects of these three parameters through comparing experimental results of the four specimens. The comparisons revealed the effects on load-carrying capacity, ductility, failure mode, deformation characteristics, and load redistribution among the remaining columns and reaction forces from surrounding structures. First, the load-carrying capacity of the specimen with slab panel dimensions of 2×2 m was around twice as great as those of specimens with slab panel dimensions of 2×3 m, while the deformation capacity of the former was much smaller than those of the latter. Second, weaker composite action decreased flexural resistance but improved ductility significantly, which was beneficial for development of tensile membrane action. Third, more flexible boundary conditions could not increase applied load beyond the stage dominated by flexure. For composite floor systems, flexural rigidity of steel columns was sufficient to support development of compressive arch action (CAA) so that additional horizontal restraints from surrounding structural members were not so beneficial at the CAA stage. However, the stiffness of additional restraints significantly affected the floor system at the catenary action stage. Finally, the typical yield pattern of composite slabs comprised negative yield lines that were formed around the perimeters and positive yield lines along the double-span girder, the double-span beam, and the paths connecting corner columns to the nearest loading points.

Specialization without Autonomy: An Informational Approach to the Development of Permanent Committees in China’s National People’s Congress

ABSTRACTThis article attempts to discover whether the National People’s Congress (NPC), China’s law-making body, is becoming more specialized and thereby losing its rubber-stamp image. The exploration of the composition of the NPC’s permanent committees demonstrates that specialization is indeed a discernible trend. More and more social elites have been co-opted onto them. The empirical results further attest to the usefulness of the information efficiency theory. Unfortunately, specialization has yet to give the Chinese legislature more autonomy. Only 30% of committee members are privileged to serve more than one five-year term. The influence of the Party-state core and the lack of membership stability have put additional restraints on the NPC’s autonomy.

High-resolution small RNA structures from exact nuclear Overhauser enhancement measurements without additional restraints

RNA not only translates the genetic code into proteins, but also carries out important cellular functions. Understanding such functions requires knowledge of the structure and dynamics at atomic resolution. Almost half of the published RNA structures have been solved by nuclear magnetic resonance (NMR). However, as a result of severe resonance overlap and low proton density, high-resolution RNA structures are rarely obtained from nuclear Overhauser enhancement (NOE) data alone. Instead, additional semi-empirical restraints and labor-intensive techniques are required for structural averages, while there are only a few experimentally derived ensembles representing dynamics. Here we show that our exact NOE (eNOE) based structure determination protocol is able to define a 14-mer UUCG tetraloop structure at high resolution without other restraints. Additionally, we use eNOEs to calculate a two-state structure, which samples its conformational space. The protocol may open an avenue to obtain high-resolution structures of small RNA of unprecedented accuracy with moderate experimental efforts.

Steric Effects on the Binding of Phosphate and Polyphosphate Anions by Zinc(II) and Copper(II) Dinuclear Complexes of m-Xylyl-bis-cyclen.

The triethylbenzene-bis-cyclen (cyclen = 1,4,7,10-tetraazacyclododecane) compound (tbmce) was designed with an imposed structural rigidity at the m-xylyl spacer to be compared to a less restrained and known parent compound (bmce). The framework of both compounds differs only in the substituents of the m-xylyl spacer. The study was centered in the differences observed in the acid-base reactions of both compounds, their copper(II) and zinc(II) complexation behaviors, as well as in the uptake of phosphate and polyphosphate anions (HPPi3-, ATP4-, ADP3-, AMP2-, PhPO42-, and HPO42-). On the one hand, the acid-base reactions showed lower values for the third and fourth protonation constants of tbmce than for bmce, suggesting that the ethyl groups of the spacer in tbmce force the two cyclen units to more conformational restricted positions. On the other hand, the stability constant values for copper(II) and zinc(II) complexes revealed that bmce is a better chelator than tbmce pointing out to additional conformational restraints imposed by the triethylbenzene spacer. The binding studies of phosphates by the dinuclear copper(II) and zinc(II) complexes showed much smaller effective association constants for the dicopper complexes. Single-crystal X-ray and computational (density functional theory) studies suggest that anion binding promotes the formation of tetranuclear entities in which anions are bridging the metal centers. Our studies also revealed the dinuclear zinc(II) complex of bmce as a promising receptor for phosphate anions, with the largest effective association constant of 5.94 log units being observed for the formation of [Zn2bmce(HPPi)]+. Accordingly, a colorimetric study via an indicator displacement assay to detect phosphates in aqueous solution found that the [Zn2bmce]4+ complex acts as the best receptor for pyrophosphate displaying a detection limit of 2.5 nM by changes visible to naked eye.

Structural analysis of glycoproteins: building N-linked glycans with Coot.

Coot is a graphics application that is used to build or manipulate macromolecular models; its particular forte is manipulation of the model at the residue level. The model-building tools of Coot have been combined and extended to assist or automate the building of N-linked glycans. The model is built by the addition of monosaccharides, placed by variation of internal coordinates. The subsequent model is refined by real-space refinement, which is stabilized with modified and additional restraints. It is hoped that these enhanced building tools will help to reduce building errors of N-linked glycans and improve our knowledge of the structures of glycoproteins.

Conformation of flexibly linked triterpene dimers by using RDC-enhanced NMR spectroscopy

Dimers of flexibly linked pentacyclic triterpene ursolic acid (UA) and its related frameworks such as asiatic acid (AA) and oleanolic acid (OA) have recently attracted significant attention due to their enhanced anti-cancer and anti-HCV activity compared to their respective monomers. Determination of conformation/inter-monomer orientation of these molecules is very important to understand their structure-activity relationship and to develop new scaffolds, which, however, is difficult through conventional NOE based solution-state NMR spectroscopy, due to lack of long-range NOEs. In the present work, we report a precise determination of conformation of two 1,2,3-triazole-linked triterpene dimer molecules, UA-AA and UA-OA, by employing one-bond CH residual dipolar couplings (RDCs) as additional long-range orientational restraints, measured in anisotropic PDMS/CDCl3 solvent medium.

Prediction of disulfide dihedral angles using chemical shifts.

Cystine residues result from the formation of disulfide bonds between pairs of cysteine residues. This cross linking of the backbone is essential for the structure and activity of peptides and proteins. The conformation of a cystine side chain can be described using five dihedral angles, χ1, χ2, χ3, χ2', and χ1', with cystines favouring certain combinations of these angles. 2D NMR spectroscopy is ideally suited for structure determination of disulfide-rich peptides, because of their small size and constrained nature. However, only limited information of the cystine side chain conformation can be determined by NMR spectroscopy, leading to ambiguity in the deduced 3D structures. Resolving accurate structures is important as disulfide-rich peptides have proven to be promising drug candidates in a number of fields, either as bioactive leads or scaffolds. Using a database of NMR chemical shifts combined with crystallographic structures, we have developed a method called DISH that uses support vector machines to predict the dihedral angles of cysteine side chains. It is able to successfully predict χ2 angles with 91% accuracy, and has improved performance over existing prediction methods for χ1 angles, with 87% accuracy. For 81% of cysteine residues, DISH successfully predicted both the χ1 and χ2 angles. By revisiting published solution structures of peptides determined using NMR spectroscopy, we assessed the impact of additional cystine dihedral restraints on the quality of 3D models. DISH improved the resolution and accuracy, highlighting the potential for improving the understanding of structure-activity relationships and rational development of peptide drugs.

Improved protein structure reconstruction using secondary structures, contacts at higher distance thresholds, and non-contacts

BackgroundResidue-residue contacts are key features for accurate de novo protein structure prediction. For the optimal utilization of these predicted contacts in folding proteins accurately, it is important to study the challenges of reconstructing protein structures using true contacts. Because contact-guided protein modeling approach is valuable for predicting the folds of proteins that do not have structural templates, it is necessary for reconstruction studies to focus on hard-to-predict protein structures.ResultsUsing a data set consisting of 496 structural domains released in recent CASP experiments and a dataset of 150 representative protein structures, in this work, we discuss three techniques to improve the reconstruction accuracy using true contacts – adding secondary structures, increasing contact distance thresholds, and adding non-contacts. We find that reconstruction using secondary structures and contacts can deliver accuracy higher than using full contact maps. Similarly, we demonstrate that non-contacts can improve reconstruction accuracy not only when the used non-contacts are true but also when they are predicted. On the dataset consisting of 150 proteins, we find that by simply using low ranked predicted contacts as non-contacts and adding them as additional restraints, can increase the reconstruction accuracy by 5% when the reconstructed models are evaluated using TM-score.ConclusionsOur findings suggest that secondary structures are invaluable companions of contacts for accurate reconstruction. Confirming some earlier findings, we also find that larger distance thresholds are useful for folding many protein structures which cannot be folded using the standard definition of contacts. Our findings also suggest that for more accurate reconstruction using predicted contacts it is useful to predict contacts at higher distance thresholds (beyond 8 Å) and predict non-contacts.

Effect of moment gradient and load height with respect to centroid on the reliability of wide flange steel beams subject to elastic lateral torsional buckling

The reliability of doubly-symmetric wide flange steel beams designed to the AISC Specification for Structural Steel Buildings subjected to elastic lateral torsional buckling was evaluated when considering variation in moment gradient and load height. The analysis considers continuous loads on spans subjected to various end moments with supports that are torsionally fixed and laterally supported, without additional intermediate restraints. Dead load, occupancy live load, and beam resistance random variables were considered. Beam lateral torsional buckling resistance was evaluated from numerical solution of a fundamental differential equation that accounts for the effect of moment gradient and load height. In some cases, it was found that use of the AISC design procedure results in significant inaccuracies for estimation of elastic lateral torsional buckling resistance, where underestimations occur in regions of reverse curvature bending and when loads are placed below the beam shear center, while large overestimations can occur when loads are placed above the beam shear center. These discrepancies result in significant variation in beam reliability. However, the use of accurate equivalent uniform moment factors can restore uniformity in notional reliability level.

Price stickiness and markup variations in market games

In this paper, we show that the Shapley–Shubik market game model with production naturally generates an equilibration mechanism that can accommodate price stickiness arising from strategic interactions of firms. Unlike New Keynesian models that show similar price stickiness results, the market game model does not require enforcing menu costs or other additional restraints on price adjustment mechanisms in order to generate price stickiness. As such, we suggest that the market game model can provide a good micro-foundation for macroeconomic analysis. We then explicitly show the relationship between a typical firm’s markup of price over marginal cost and its market share.

The development of the buckling restrained braces with new end restrains

This paper presents an experimental investigation of buckling restrained braces (BRBs) with new end restraints and casing members (CMs). The component tests for ten BRBs with CMs consisting of concrete-filled steel tube (unbonded), plain concrete, plain concrete wrapped with Fiber-Reinforced Polymer (FRP), reinforced concrete and a built-up section were tested up to a core plate (CP) strain of 2.0%. In unbonded BRBs, an unrestrained part is usually available on the CP. This part may be a candidate for buckling during cyclic excursions. Hence both ends of the BRBs at the unrestrained part of the CP need to be restrained more effectively. The innovations of BRBs in the present study were that additional end restraints were added at the unrestrained part of the CP at both ends, isolation material was employed, and a more economical CM was used. These new end restraints consisted of hollow steel sections and steel plates welded to each other and were attached to the CM. The testing of the improved BRBs indicated that the cyclic performance of the BRBs was satisfactory up to a CP strain of 2.0%. The energy dissipation capacity of the BRBs was found to be significantly dependent on compression strength adjustment factor, β, and strain hardening adjustment factor, ω. Consequently, the improved BRBs with sufficient stiffness to resist out-of-plane buckling at both ends have acceptable cyclic performance according to the test results. Furthermore, the connection details namely slip critical, isolation materials, and their application techniques have also been investigated for the improved BRB design in this study.

The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ω-Hydroxylation

P450 family 4 fatty acid ω-hydroxylases preferentially oxygenate primary C-H bonds over adjacent, energetically favored secondary C-H bonds, but the mechanism explaining this intriguing preference is unclear. To this end, the structure of rabbit P450 4B1 complexed with its substrate octane was determined by X-ray crystallography to define features of the active site that contribute to a preference for ω-hydroxylation. The structure indicated that octane is bound in a narrow active-site cavity that limits access of the secondary C-H bond to the reactive intermediate. A highly conserved sequence motif on helix I contributes to positioning the terminal carbon of octane for ω-hydroxylation. Glu-310 of this motif auto-catalytically forms an ester bond with the heme 5-methyl, and the immobilized Glu-310 contributes to substrate positioning. The preference for ω-hydroxylation was decreased in an E310A mutant having a shorter side chain, but the overall rates of metabolism were retained. E310D and E310Q substitutions having longer side chains exhibit lower overall rates, likely due to higher conformational entropy for these residues, but they retained high preferences for octane ω-hydroxylation. Sequence comparisons indicated that active-site residues constraining octane for ω-hydroxylation are conserved in family 4 P450s. Moreover, the heme 7-propionate is positioned in the active site and provides additional restraints on substrate binding. In conclusion, P450 4B1 exhibits structural adaptations for ω-hydroxylation that include changes in the conformation of the heme and changes in a highly conserved helix I motif that is associated with selective oxygenation of unactivated primary C-H bonds.

Coordination Equilibrium, Price Stickiness, and Markup Variations in Market Games

In this paper, we show that the Shapley-Shubik market game model with production naturally generates an equilibration mechanism that can lead to indeterminate coordination equilibria and can accommodate price stickiness arising from strategic interactions of firms. Unlike New Keynesian models that show similar price stickiness results, the market game model does not require enforcing menu costs or other additional restraints on price adjustment mechanisms in order to generate price stickiness. As such, we suggest that the market game model can provide a good micro-foundation for macroeconomic analysis. We then explicitly show the relationship between a typical firm's markup of price over marginal cost and its market share.

Hybrid Applications of Solution Scattering to Aid Structural Biology.

Biomolecular applications of solution X-ray and neutron scattering (SAXS and SANS, respectively) started in late 1960s - early 1970s but were relatively limited in their ability to provide a detailed structural picture and lagged behind what became the two primary methods of experimental structural biology - X-ray crystallography and NMR. However, improvements in both data analysis and instrumentation led to an explosive growth in the number of studies that used small-angle scattering (SAS) for investigation of macromolecular structure, often in combination with other biophysical techniques. Such hybrid applications are nowadays quickly becoming a norm whenever scattering data are used for two reasons. First, it is generally accepted that SAS data on their own cannot lead to a uniquely defined high-resolution structural model, creating a need for supplementing them with information from complementary techniques. Second, solution scattering data are frequently applied in situations when a method such NMR or X-ray crystallography cannot provide a satisfactory structural picture, which makes these additional restraints highly desirable. Maturation of the hybrid bio-SAS approaches brings to light new questions including completeness of the conformational space sampling, model validation, and data compatibility.