Additional Restraints Research Articles

Experimental study on seismic performance of spatial and planar hoop-head mortise and tenon timber joints in historical architecture

This paper aims to present an experimental study investigating the seismic behaviour of spatial and planar hoop-head mortise and tenon (MT) timber joints in traditional Guangfu Ancestral Halls. The unique design of the external hoop head provides additional restraints for the tenon and mortise in these joints, endowing them with enhanced pull-out resistance and increased structural integrity compared to other types of MT connections commonly used in traditional timber-framed structures. The research commenced with material testing to determine the mechanical properties of Merbau wood used in this timber frame construction. Reversed cyclic loading tests were then performed on two planar and two spatial hoop-head MT timber joints. The experimental results demonstrated the favourable seismic performance of the hoop-head MT joints. All specimens failed within the MT joint area, exhibiting different failure modes based on varying connection configurations and tenon sizes. Furthermore, the spatial MT joints showed lower stiffness and bearing capacities compared to the planar MT joints of equivalent size. Their hysteresis curves, envelope curves, and cyclic stiffness degradation curves displayed greater asymmetry. These discrepancies can be attributed to the reduction in tenon sections necessary to accommodate the overlapping beams within the spatial joint core area.

Adverse effects of unilateral transfemoral amputation on para-alpine sit skiers and mitigation methods

ObjectivesThis study aimed to evaluate the adverse effects of unilateral transfemoral amputation on neuromuscular and kinematic parameters in alpine sit skiers, and to determine if additional restraints on the human–bucket interface could help mitigate the effects. DesignCross-sectional, repeated measures study. MethodsSimulated skiing tests were conducted indoors involving 10 skiers with unilateral transfemoral amputation and 10 able-bodied participants. A Paralympic silver medalist performed slalom skiing tests on snow. These tests were conducted with and without additional strapping on the residual limb. Surface electromyography of trunk muscles and athletic performance was measured, and the asymmetry index was calculated. ResultsAthletes were significantly dependent on muscle activation on the dominant side (asymmetry index = 7.8 %–28.3 %, p < 0.05). Worse athletic performance to the dominant side was found based on inclination angles of the indoor board (asymmetry index = −9.8 %, p = 0.014) and outdoor sit ski (−11.1 %, p = 0.006), and distance to the gate poles during skiing turns (18.6 %, p < 0.001). After using additional restraints, the above asymmetry index declined significantly (asymmetry index < 4.5 %, p < 0.05). Furthermore, athletic performance was significantly improved on both body sides by 11.1 %–30.7 % (p < 0.05). ConclusionsUnilateral transfemoral amputation caused the dependence on the trunk muscles of the dominant side and the corresponding unilateral poor performance in athletes. Adjusting restraints in the human–equipment interface by additional strapping could mitigate the asymmetry issues and improve athletic performance.

Accelerating and Automating the Free Energy Perturbation Absolute Binding Free Energy Calculation with the RED-E Function.

The accurate prediction of the binding affinities between small molecules and biological macromolecules plays a fundamental role in structure-based drug design, which is still challenging. The free energy perturbation-based absolute binding free energy (FEP-ABFE) approach has shown potential in its reliability. To correctly calculate the energy related to the ligand being restrained by the receptor, additional restraints between the ligand and the receptor are needed. However, determining the restraint parameters for individual ligands empirically is too trivial to be automated, and usually gives rise to numerical instabilities, which set back the applications of FEP-ABFE. To address these issues, we derived the analytical expression for the probability distribution of energy differences, P(ΔU), during the process of restraint addition, which is called the RED-E (restraint energy distribution at equilibrium position) function. Simulations indicated that the RED-E function can accurately describe P(ΔU) when restraints are added at the equilibrium position. Based on the RED-E function, an automatic restraint selection method was proposed to select the best restraint. With this method, there is a high phase-space overlap between the free and restrained states, such that using a 2-λ perturbation can accurately calculate the free energy of the restraint addition, which is a nearly 6 times acceleration compared with current widely used 12-λ perturbation method. The RED-E function gives insight into the non-Gaussian behavior of the sampled P(ΔU) in certain FEP processes in an analytical way. The highly automated and accelerated restraint selection also makes it possible for the large-scale application of FEP-ABFE in real drug discovery practices.

RNAJP: enhanced RNA 3D structure predictions with non-canonical interactions and global topology sampling.

RNA 3D structures are critical for understanding their functions. However, only a limited number of RNA structures have been experimentally solved, so computational prediction methods are highly desirable. Nevertheless, accurate prediction of RNA 3D structures, especially those containing multiway junctions, remains a significant challenge, mainly due to the complicated non-canonical base pairing and stacking interactions in the junction loops and the possible long-range interactions between loop structures. Here we present RNAJP ('RNA Junction Prediction'), a nucleotide- and helix-level coarse-grained model for the prediction of RNA 3D structures, particularly junction structures, from a given 2D structure. Through global sampling of the 3D arrangements of the helices in junctions using molecular dynamics simulations and in explicit consideration of non-canonical base pairing and base stacking interactions as well as long-range loop-loop interactions, the model can provide significantly improved predictions for multibranched junction structures than existing methods. Moreover, integrated with additional restraints from experiments, such as junction topology and long-range interactions, the model may serve as a useful structure generator for various applications.

Safety and effectiveness of benzodiazepines and antipsychotics for agitation in older adults in the emergency department

PurposeTo examine the safety and effectiveness of benzodiazepines (BZD) as compared to antipsychotics for the management of acute agitation in older adults in the emergency department (ED). Basic proceduresRetrospective observational cohort study of 21 EDs across four states in the US, including adults ≥60 years old who received either BZD or antipsychotics for acute agitation in the ED and subsequently were admitted to the hospital. Safety was measured as presence of adverse events: respiratory depression, cardiovascular effects, extrapyramidal side effects, or a fall during hospitalization. Effectiveness was measured as indicators of treatment failure: need for additional medication, one-to-one observation, or physical restraints following initial medication administration. Proportions and odds ratios with 95% confidence intervals (CI) were calculated. Univariable and multivariable logistic regression were used to assess the association between potential risk factors and for efficacy and safety endpoints. Main findingsA total of 684 patients were included (63.9% received a BZD and 36.1% an antipsychotic). There was no difference in the incidence of adverse events between groups (20.6% vs 14.6%, difference 6.0%, 95% CI -0.2% to 11.8%), but there was a higher intubation rate in the BZD group (2.7% vs 0.4%, difference 2.3%). There were more treatment failures in the antipsychotic group for the composite primary efficacy endpoint (94.3% vs 87.6%, difference 6.7%, 95% CI 2.5% to 10.9%). This appears to have been driven by the need for 1:1 observation; sensitivity analysis excluding 1:1 observation in the composite outcome demonstrated no significant difference with a failure rate of 38.5% in the antipsychotic group and 35.2% in the benzodiazepine group. Principal conclusionsOverall there are high rates of treatment failure among agitated older adults receiving pharmacological treatment for agitation in the emergency department. The optimal selection of pharmacological treatment for agitation in older adults should be made considering patient-specific factors that could increase the risk of adverse effects or treatment failure.

Overcoming hysteresis in ligand binding potential of mean force calculations.

Accurate estimation of ligand binding free energy and elucidation of the corresponded binding pathway are key challenges in computational drug design. Umbrella sampling (US) molecular dynamic simulations are widely used to tackle these challenges; however, they frequently suffer from massive hysteresis between the ligand binding and unbinding pathways, limiting the applicability of US for ligand-protein interaction studies. The convergence of US is affected by the complexity of the system, the selection of reaction coordinate(s), simulation time, and by the selection of additional restraints.

Parametrization of Force Field Bonded Terms under Structural Inconsistency.

Parametrization of the bonded part of molecular mechanics (MM) force fields (FFs) is typically done by fitting reference quantum mechanical (QM) energies or forces of representative structures. FFs for small molecules are constructed in incremental parametrization procedures, where parameters developed previously are retained for novel molecules, followed by optimization of missing, not previously optimized parameters. Equilibrium QM and MM geometries of molecules can deviate due to parameters transferred from existing molecules in the FF. In this work, we demonstrate that conventional parametrization methods based on fitting QM energies and/or forces to derive parameters for bond and angle terms produce largely suboptimal force constants when MM and QM equilibrium structures deviate. We further developed and tested a new method to derive CHARMM FF parameters based on the potential energy surface scans where a structural deviation between QM and MM optimized geometries is explicitly allowed during parametrization. The test of the new method was performed on a diverse set of 32 molecules. The results show that without any need for additional restraints, the new method produces robust and largely transferable parameters for bond and angle terms. The new method also improves the agreement for the normal modes for all molecules in the test set, reducing the average error in the reproduction of QM normal mode frequencies from 9.5% computed with CGenFF parameters to 6.8% computed with the new parameters. The new method will allow parametrization of molecules under structural deviations, common for force fields for small molecules, producing robust and transferable parameters.

Mechanical Stability of Ribonuclease A Heavily Depends on the Redox Environment.

Disulfide bonds are covalent bonds that connect nonlocal fragments of proteins, and they are unique post-translational modifications of proteins. They require the oxidizing environment to be stable, which occurs for example during oxidative stress; however, in a cell the reductive environment is maintained, lowering their stability. Despite many years of research on disulfide bonds, their role in the protein life cycle is not fully understood and seems to strictly depend on a system or process in which they are involved. In this article, coarse-grained UNited RESidue (UNRES), and all-atom Assisted Model Building with Energy Refinement (AMBER) force fields were applied to run a series of steered molecular dynamics (SMD) simulations of one of the most studied, but still not fully understood, proteins—ribonuclease A (RNase A). SMD simulations were performed to study the mechanical stability of RNase A in different oxidative–reductive environments. As disulfide bonds (and any other covalent bonds) cannot break/form in any classical all-atom force field, we applied additional restraints between sulfur atoms of reduced cysteines which were able to mimic the breaking of the disulfide bonds. On the other hand, the coarse-grained UNRES force field enables us to study the breaking/formation of the disulfide bonds and control the reducing/oxidizing environment owing to the presence of the designed distance/orientation-dependent potential. This study reveals that disulfide bonds have a strong influence on the mechanical stability of RNase A only in a highly oxidative environment. However, the local stability of the secondary structure seems to play a major factor in the overall stability of the protein. Both our thermal unfolding and mechanical stretching studies show that the most stable disulfide bond is Cys65–Cys72. The breaking of disulfide bonds Cys26–Cys84 and Cys58–Cys110 is associated with large force peaks. They are structural bridges, which are mostly responsible for stabilizing the RNase A conformation, while the presence of the remaining two bonds (Cys65–Cys72 and Cys40–Cys95) is most likely connected with the enzymatic activity rather than the structural stability of RNase A in the cytoplasm. Our results prove that disulfide bonds are indeed stabilizing fragments of the proteins, but their role is strongly redox environment-dependent.

Unintended Consequences of Pandemic Management Strategies on Residents and Family in One Long-term Care Home in British Columbia: A Patient-Supported Qualitative Study.

Background and ObjectivesIn March 2020, pandemic management strategies were mandated across long-term care homes in British Columbia, Canada, to control the effects of COVID-19. This study describes and contextualizes the impact of visitation, infection prevention and control, and staffing strategies on the perceived health and well-being of residents and families.Research Design and MethodsThis interpretive description study was part of a larger mixed-methods study at a publicly funded not-for-profit long-term care home in British Columbia, Canada. Eleven family members and 10 residents were interviewed between October and December 2020, and resident and family partners participated in a steering committee throughout all stages of the research.FindingsEarly pandemic management strategies had an adverse impact on the perceived health and well-being of families and residents. Visitation restrictions eliminated care routinely provided by families and prevented in-person communication between residents, families, and care providers. Other infection prevention and control strategies isolated residents; group enrichment programs were stopped, and lockdowns created a perception of incarceration. Donning and doffing personal protective equipment took time away from staff–resident interactions and the single-site order reduced staff numbers, placing additional time restraints on residents’ care.Discussion and ImplicationsUnintended adverse consequences of pandemic management strategies demonstrate the risks of creating policies based on a medicalized definition of health. Clear lines of communication are vital to increase a sense of control for families and residents. Elimination of care provided by families and paid companions exposed a gap in Canada’s public long-term care system. This care gap raises concerns about equitable care access for residents without families or financial means to pay for additional care.

О построении циркулянтных матриц, связанных с MDS-матрицами

The objective of this paper is to suggest a method of the construction of circulant matrices, which are appropriate for being MDS (Maximum Distance Separable) matrices utilising in cryptography. Thus, we focus on designing so-called bi-regular circulant matrices, and furthermore, impose additional restraints on matrices in order that they have the maximal number of some element occurrences and the minimal number of distinct elements. The reason to construct bi-regular matrices is that any MDS matrix is necessarily the bi-regular one, and two additional restraints on matrix elements grant that matrix-vector multiplication for the samples constructed may be performed efficiently. The results obtained include an upper bound on the number of some element occurrences for which the circulant matrix is bi-regular. Furthermore, necessary and sufficient conditions for the circulant matrix bi-regularity are derived. On the basis of these conditions, we developed an efficient bi-regularity verification procedure. Additionally, several bi-regular circulant matrix layouts of order up to 31 with the maximal number of some element occurrences are listed. In particular, it appeared that there are no layouts of order 32 with more than 5 occurrences of any element which yield a bi-regular matrix (and hence an MDS matrix).

Proceduralni aspekti ustavne revizije u oblasti pravosuđa i ograničenje nadležnosti Narodne Skupštine

The subject of this paper is an analysis of the procedure of the amendments to the "Mitrovdan" Constitution from the perspective of adherence to the procedure and protection of the constitutional continuity. Considering the multivalent effect of the constitutional revision on the strenghtening of the constitutional order, constitutional culture, and rule of law in an unique legal-political moment immediately following the proclamation of the Act on Amendments to the Constitution, but prior to the enactment of the set of judicial laws, the study aims to analyse whether the procedure of the constitutional amendment contributed to the furtherance of the constitutional democracy. The scope of the study is limited to a procedure, the sequence of formal acts following a prescribed procedure for the constitutional amendment, while the subject of the amendments is reflected upon only as it is necessary for understanding both the essence and context of the matter. It is indisputable that by adopting 29 amendments to the "Mitrovdan" Constitution and guaranteeing the independence of the judiciary and prosecution there was made an important step in the process of overcoming tensions between both the form and substance of the constitutional political culture. But, at the same time, there were some additional restraints of the authorities of the National Assembly. The process of improving the constitutional democracy, preserving the constitutional continuity, and building the constitutional culture of Serbia is analysed from a historical perspective, with a special reference to the procedure for adopting the 2006 Constitution. The methodological, historical-comparative approach is completed by analyzing the comments and interpretations of certain constitutional acts, laws, and bylaws. The additional value of the analysis stems from the methodological facts being interpreted from the perspective of a fifteen-year experience as a Member of Parliament, and a direct participant in the constitutional review process as a member of the Committee on Constitutional Affairs and Legislation of the National Assembly.

Mechanistic Modelling of Slow and Fast NHEJ DNA Repair Pathways Following Radiation for G0/G1 Normal Tissue Cells.

Simple SummaryWhen cells are irradiated, their DNA can become damaged, this causes different types of repair processes to try and fix the DNA breaks. One of the most lethal types of DNA damage is double-strand breaks (DSBs). This work models the most used DSB repair process called Non-Homologous End Joining (NHEJ) and includes both its resection-independent and resection-dependent pathways. The models produced are benchmarked against experimental normal and deficient cell-types across a wide range of radiation qualities. We compare two approaches of modelling, the first is where the DSBs can repair in parallel and the second is where the DSBs repair is entwined. We find that it is necessary to consider both the resection-independent and resection-dependent pathways as entwined to produce a model which robustly matches experimental work. Through better modelling of NHEJ repair, it can improve our understanding of radiation response which has potential in biological optimisation for radiotherapy.Mechanistic in silico models can provide insight into biological mechanisms and highlight uncertainties for experimental investigation. Radiation-induced double-strand breaks (DSBs) are known to be toxic lesions if not repaired correctly. Non-homologous end joining (NHEJ) is the major DSB-repair pathway available throughout the cell cycle and, recently, has been hypothesised to consist of a fast and slow component in G0/G1. The slow component has been shown to be resection-dependent, requiring the nuclease Artemis to function. However, the pathway is not yet fully understood. This study compares two hypothesised models, simulating the action of individual repair proteins on DSB ends in a step-by-step manner, enabling the modelling of both wild-type and protein-deficient cell systems. Performance is benchmarked against experimental data from 21 cell lines and 18 radiation qualities. A model where resection-dependent and independent pathways are entirely separated can only reproduce experimental repair kinetics with additional restraints on end motion and protein recruitment. However, a model where the pathways are entwined was found to effectively fit without needing additional mechanisms. It has been shown that DaMaRiS is a useful tool when analysing the connections between resection-dependent and independent NHEJ repair pathways and robustly matches with experimental results from several sources.

Structure of the ATP‐free Mre11‐Rad50 DNA damage repair complex bound to DNA substrates

DNA double strand breaks, where both strands of the double-stranded DNA are broken, are among the most harmful forms of DNA damage because an intact template is not present for repair. The Mre11-Rad50–Nbs1 (Xrs2 in yeast) complex plays a central and critical role in the detection and repair of DSBs and is conserved in all kingdoms of life, as Mre11Rad50 (MR) in prokaryotes and as MRN/X in eukaryotes. Although there are some structural models available for MR complex bound to DNA substrates, there are limitations in these structures. Therefore, our goal is to examine the MR complex bound to different DNA substrates using an integrated structural biology approach. Using uniformly deuterated, 13 CH -methyl labeled samples, we measured distance restraints for the ~120 kDa P. furiosus MR complex bound to spin-labeled double-stranded or single-stranded DNA substrates 3 via paramagnetic relaxation enhancements (PREs). Additional distance and global restraints were measured from Luminescence Resonance Energy Transfer (LRET) and Small Angle X-ray Scatter (SAXS) experiments. These data then served as restraints for calculating structural models using multi-body docking implemented in HADDOCK. Our models revealed novel modes of DNA binding for the ATP-free MR complex and provide the first depiction of a single-stranded DNA-bound complex. We tested our models through biochemical activity assays to determine the effect of disruption of key interactions, via mutation, in our models.

Setting up Reference Variants to Comply With Current Boundary Settings in Finite Set Model Predictive PMSM Control

Finite control set model predictive control (FCS-MPC), especially the reference-voltage-vector-based model predictive control (MPC) (RVV-MPC), considers a limited set of input vectors. However, the control performance is not satisfactory enough due to a severe lack of voltage input variants. In particular, additional restraints, such as limiting the currents within setting boundaries, could not be supported with RVV-MPC. In this article, to truly comply with the boundary settings, a reference variant FCS-MPC approach is introduced. In the proposed strategy, reference currents are considered under the maximum torque per current (MTPC) control. As boundary settings could not be supported in RVV-MPC, it is better to search for the optimal current reference that is feasible within the imposed boundaries. Therefore, rather than fixing the current reference, a well-considered neighborhood of the MTPC reference is introduced, and these variants are processed, resulting in a set of optimal candidate inputs. As multiple, but still finite, optimal solutions become possible, additional efforts can be made within the objective function to restrain the current and reduce the torque ripple. The size of the current reference space can be automatically adapted to generate current references that correspond to truly bounded current vectors. A trade-off is then made between control performance and meeting the boundary settings.

A Pipeline for Integrative Structural Modelling of Very Complex Molecular Complexes

Although electron microscopy (EM) allows determining the structure of macromolecular complexes at near-atomic resolution, high resolution is still difficult to achieve for complexes that are flexible, heterogenous, or imaged in cells with cryo-electron tomography. A state-of-the-art methodology to obtain structures of such complexes is integrative structural modelling, which combines data from complementary techniques, such as X-ray crystallography, EM, NMR, SAXS or cross-linking mass spectrometry. The current integrative modelling software, however, is either limited to complexes of simple architectures or requires custom modifications, and thus a major expertise, when applied to more complex architectures. Thus, we developed a versatile modelling pipeline for integrative modelling of protein complexes with very complex architecture in an accessible and efficient way. Our pipeline is implemented using Integrative Modeling Platform library, on top of which it implements custom algorithms for efficient sampling of the conformational space, versatile configuration language and graphical interface for input preparation, modelling and analysis, and additional restraints. Flexible and symmetrical modeling is also supported. The pipeline enables modeling of very complex architectures, for example, with multiple symmetries or with EM maps for alternative conformational states. I will present the pipeline and how we applied it to model complexes ranging from the 1 MDa Elongator to the 110 MDa human nuclear pore complex (NPC). For the Elongator complex, I will present how our published model agrees with the subsequent high-resolution cryo-EM structure. For the NPC, I will show our most recent results of how the pipeline allowed building structural models of NPCs from yeast S. cerevisiae and S. pombe, including a novel multi-state modelling protocol for modelling based on multiple EM maps simultaneously. The new NPC models reveal surprisingly different architectures and depict conformational changes of NPCs in response to cellular states.

Structure of the ATP-Free Mre11-Rad50 DNA Damage Repair Complex Bound to DNA Substrates

DNA double strand breaks, where both strands of the double-stranded DNA are broken, are among the most harmful forms of DNA damage because an intact template is not present for repair. The Mre11-Rad50–Nbs1 (Xrs2 in yeast) complex plays a central and critical role in the detection and repair of DSBs and is conserved in all kingdoms of life, as Mre11- Rad50 (MR) in prokaryotes and as MRN/X in eukaryotes. Although there are some structural models available for MR complex bound to DNA substrates, there are limitations in these structures. Therefore, our goal is to examine the MR complex bound to different DNA substrates using an integrated structural biology approach. Using uniformly deuterated, 13CH3-methyl labeled samples, we measured distance restraints for the ∼120 kDa P. furiosus MR complex bound to spin-labeled double-stranded or single-stranded DNA substrates via paramagnetic relaxation enhancements (PREs). Additional distance and global restraints were measured from Luminescence Resonance Energy Transfer (LRET) and Small Angle X-ray Scatter (SAXS) experiments. These data then served as restraints for calculating structural models using multi-body docking implemented in HADDOCK. Our models revealed novel modes of DNA binding for the ATP-free MR complex and provide the first depiction of a single-stranded DNA-bound complex. We tested our models through biochemical activity assays to determine the effect of disruption of key interactions, via mutation, in our models.

Parametrizing the Spatial Dependence of 1H NMR Chemical Shifts in π-Stacked Molecular Fragments.

Most recently a renewed interest in several areas has arisen in factors governing the 1H NMR chemical shift (1H CS) of protons in aromatic systems. Therefore, it is important to describe how 1H CS values are affected by π-stacking intermolecular interactions. The parametrization of radial and angular dependences of the 1H CS is proposed, which is based on conventional gauge-independent atomic orbital (GIAO) calculations of explicit molecular fragments. Such a parametrization is exemplified for a benzene dimer with intermonomer vertical and horizontal distances which are in the range of values often found in crystals of organic compounds. Results obtained by the GIAO calculations combined with B3LYP and MP2 methods were compared, and revealed qualitatively the same trends in the 1H CS data. The parametrization was found to be quantitatively correct for the T-shaped benzene dimers, and its limitations were discussed. Parametrized 1H CS surfaces should become useful for providing additional restraints in the search of site-specific information through an analysis of structurally induced 1H CS changes.

Seating preferences in highly automated vehicles and occupant safety awareness: A national survey of Chinese perceptions

Objective: The potential challenge for providing occupant protection accompanying seating preferences is an essential safety prerequisite for highly automated vehicle (HAV) popularization. This research is aimed toward identifying Asia-specific individualized seating preferences in HAVs and occupant safety awareness via a national survey in China.Methods: An online questionnaire survey was performed to investigate seating preferences (i.e., sitting posture, seating orientation, and position) and occupant safety awareness (i.e., seat belt usage and receptiveness to extended or additional restraints beyond the conventional three-point seat belt). We assessed whether perceptions were modulated by individual characteristics via bivariate and correlation analyses. The possibility of wearing seat belts was estimated by binary logistic regression.Results: The final survey data set includes 1,018 respondents after a rigorous validity check (response rate: 59.2%). The results show that preferred sitting postures and seating orientation were significantly associated with sociodemographic characteristics (e.g., gender, age, city tier) (p < 0.05). The rear seat was preferred in both the conventional (65.6%) and “face-to-face mode” seating configurations (77.6%), largely due to the fact that customers subjectively viewed it as being safer than sitting in a front seat in case of collisions. Despite the current trend of an increasing usage rate of seat belts, 48.5% of respondents preferred to be unrestrained in rear seats, especially for the subgroups who were from less developed cities and with a higher usage rate of public transport (p < 0.01). Low receptiveness to extended restraint and high comfort requirements were confirmed for the young, high-frequency road users, and for those who were from developed areas (p < 0.05).Conclusions: Diversified and specific seating preferences of Chinese occupants were identified facing emerging use of HAVs. Next generation occupant protection systems shall be adapted to account for the individualized expectations and needs on seating designs from certain population groups. Balanced restraint design between safety and comfort was required to exceed the existing strong dependence on exogenous causes of restraint use (e.g., legal restrictions) in Asia.

NMR “Crystallography” of Membrane Proteins Aligned in Native-Like Bilayers

Oriented-sample (OS) solid-state NMR can be used to determine structures of membrane proteins without crystallization and use of cryogenic temperatures. Here, the orientationally dependent dipolar couplings and chemical shifts provide direct input for structure calculations. However, so far only the 1H-15N dipolar couplings and 15N chemical shifts have been routinely assessed in 15N labeled protein samples. While highly efficient for determining the tilt angles of short α-helical peptides, these two measurements alone are likely insufficient for determining protein structures of arbitrary topology. Therefore, determination of tertiary structures of membrane proteins by OS NMR requires measurements of additional angular restraints. Here we have developed a new experimental triple-resonance NMR technique, which was applied to uniformly doubly (15N, 13C) labeled Pf1 coat protein reconstituted in magnetically aligned DMPC/DHPC bicelles. The previously inaccessible 1Hα-13Cα dipolar couplings, which represent powerful chiral angular restraints, have been measured for the resolved NMR resonances of the transmembrane domain of Pf1. Inclusion of the third restraint makes it possible to determine the torsion angles Φ and Ψ between the adjacent peptide planes without assuming α-helical structure a priori. Using our recently developed structure calculation algorithm [1], simultaneous fitting of three restraints per peptide plane has resulted in families of structures which have subsequently been filtered using various Rosetta scoring functions including the energetics within the membrane environment, hydrogen bonding, and potential steric clashes. Such structural validation protocol has led to a consensus α-helical transmembrane structure for Pf1 coat protein, albeit obtained in a de novo fashion, thus greatly enhancing utility of OS NMR as a “crystallography” in near-native membrane environments. [1]. Lapin, J. and Nevzorov A.A., Validation of protein backbone structures calculated from NMR angular restraints using Rosetta, J. Biomol. NMR, 2019; 73(5):229-244.

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

AbstractIn 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 1H–15N dipolar couplings and 15N chemical shifts have been routinely assessed in oriented 15N‐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 (15N, 13C)‐labeled Pf1 coat protein in magnetically aligned DMPC/DHPC bicelles. The previously inaccessible 1Hα–13Cα 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.