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At least 811 records · Page 45

Flight Test Determined Aerodynamics Force and Moment Characteristics of the X-43A Research Vehicle at Mach 7.0

The second flight of the HYPER-X Program afforded a unique opportunity to determine the aerodynamic force and moment characteristics of an airframe integrated scramjet powered aircraft in hypersonic flight. These data were gathered via a repeated series of pitch, yaw, and roll doublets, frequency sweeps, and pull-up/push-over maneuvers performed throughout the X-43A cowl-closed descent phase. The subject flight research maneuvers were conducted in a Mach number range of 6.8 to 0.95 at altitudes from 92,000 ft to sea level. In this flight regime, the dynamic pressure varied from 1300 psf to 400 psf with angle-of-attack ranging from 0 deg to 14 deg. The flight-extracted aerodynamics were compared with pre-flight predictions based on wind tunnel test data. The X-43A flight-derived axial force was found to be 10 to 15 percent higher than prediction. Under-predictions of similar magnitude were observed for the normal force. For Mach numbers greater than 4, the X-43A flight-derived stability and control characteristics resulted in larger than predicted static margins, with the largest discrepancy approximately 5-inches forward along the X(CG) at Mach 6. This would result in less static margin in pitch. The X-43A predicted lateral-directional stability and control characteristics matched well with flight data when allowance was made for the high uncertainty in angle-of-sideslip.

Davis, Mark C.↗

Pterodactyl: The Development and Performance of Guidance Algorithms for a Mechanically Deployed Entry Vehicle

Pterodactyl is a NASA Space Technology Mission Directorate (STMD) project focused on developing a design capability for optimal, scalable, Guidance and Control (G&C) solutions that enable precision targeting for Deployable Entry Vehicles (DEVs). This feasibility study is unique in that it focuses on the rapid integration of targeting performance analysis with structural & packaging analysis, which is especially challenging for new vehicle and mission designs. This paper will detail the guidance development and trajectory design process for a lunar return mission, selected to stress the vehicle designs and encourage future scalability. For the five G&C configurations considered, the Fully Numerical Predictor-Corrector Entry Guidance (FNPEG) was selected for configurations requiring bank angle guidance and FNPEG with Uncoupled Range Control (URC) was developed for configurations requiring angle of attack and sideslip angle guidance. Successful G&C configurations are defined as those that can deliver payloads to the intended descent and landing initiation point, while abiding by trajectory constraints for nominal and dispersed trajectories.

Johnson, Breanna J.↗

ModuleOT: A Hardware Security Module for Operational Technology: Preprint

With increasing penetration levels of distributed energy resources (DERs) on the distribution grid, as well as new technological advancements in the cyber space, new cyberattack vectors are being introduced, and the available attack surface is constantly increasing. Despite this increasing risk, the standard IEEE 1547-2018 does not yet recommend cybersecurity measures for DERs. To address this and to better protect data on the distribution grid - from the standpoints information security as well as operational security - ModuleOT has been developed. The module aims to significantly reduce cyberattack vectors by improving data privacy for user applications. This is accomplished by performing the core functions of encryption, authentication, authorization, certificate management, and user access control. The module integrates a custom security application with hardware cryptographic acceleration. The application secures all communications using Transmission Control Protocol over Internet Protocol (TCP/IP). These include the three most commonly used communications protocols for power systems information exchange: Modbus, Distributed Network Protocol 3 (DNP3), and Smart Energy Profile 2.0 (SEP2.0). These three protocols are also supported by IEEE 1547-2018 for all DER devices. This paper tests the data encryption/decryption feature on a physical networking test bed with emulated Modbus devices reporting grid data and presents the results.

24 POWER TRANSMISSION AND DISTRIBUTION↗

Graph interpolating activation improves both natural and robust accuracies in data-efficient deep learning

Improving the accuracy and robustness of deep neural nets (DNNs) and adapting them to small training data are primary tasks in deep learning (DL) research. In this paper, we replace the output activation function of DNNs, typically the data-agnostic softmax function, with a graph Laplacian-based high-dimensional interpolating function which, in the continuum limit, converges to the solution of a Laplace–Beltrami equation on a high-dimensional manifold. Furthermore, we propose end-to-end training and testing algorithms for this new architecture. The proposed DNN with graph interpolating activation integrates the advantages of both deep learning and manifold learning. Compared to the conventional DNNs with the softmax function as output activation, the new framework demonstrates the following major advantages: First, it is better applicable to data-efficient learning in which we train high capacity DNNs without using a large number of training data. Second, it remarkably improves both natural accuracy on the clean images and robust accuracy on the adversarial images crafted by both white-box and black-box adversarial attacks. Third, it is a natural choice for semi-supervised learning. This paper is a significant extension of our earlier work published in NeurIPS, 2018. For reproducibility, the code is available at https://github.com/BaoWangMath/DNN-DataDependentActivation .

Mathematics↗

PMU Data Integrity Evaluation through Analytics on a Virtual Test-Bed

Power systems are rapidly becoming populated by phasor measurement units (PMUs) in ever increasing numbers. PMUs are critical components of today s energy management systems, designed to enable near real-time wide area monitoring and control of the electric power system. They are able to measure highly accurate bus voltage phasors as well as branch current phasors incident to the buses at which PMUs are equipped. Synchrophasor data is used for applications varying from state estimation, islanding control, identifying outages, voltage stability detection and correction, disturbance recording, and others. However, PMU-measured readings may suffer from errors due to meter biases or drifts, incorrect configurations, or even cyber-attacks. Furthermore, the testing of early PMUs showed a large disparity between the reported values from PMUs provided by different manufacturers, particularly when frequency was off-nominal, during dynamic events, and when harmonic/inter-harmonic content was present. Detection and identification of PMU gross measurement errors are thus crucial in maintaining highly accurate phasor readings throughout the system. In this paper, we present our work in conducting analytics to determine the trustworthiness and worth of the PMU readings collected across an electric network system. By implementing the IEEE 118 bus test case on a virtual test bed (VTB) , we are able to emulate PMU readings (bus voltage and branch current phasors in addition to bus frequencies) under normal and abnormal conditions using (virtual) PMU sensors deployed across major substations in the network. We emulate a variety of failures such as bus, line, transformer, generator, and/or load failures. Data analytics on the voltage phase angles and frequencies collected from the PMUs show that specious (or compromised) PMU device(s) can be identified through abnormal behaviour by comparing the trend of its frequency and phase angle reading with the ensemble of all other PMU readings in the network. If the reading trend of a particular PMU deviates from the weighted average of the reading trends of other PMUs at nearby substations, then it is likely that the PMU is malfunctioning. We assign a weight to each PMU denoting how electric-topology-wise close it is from where the PMU under consideration is located. The closer a PMU is, the higher the weight it has. To compute the closeness between two nodes in the power network, we employ a form of the resistance distance metric. It computes the electrical distance by taking into consideration the underlying topology as well as the physical laws that govern the electrical connections or flows between the network components. The detection accuracy of erroneous PMUs should be improved by employing this metric. We present results to validate the proposed approach. We also discuss the effectiveness of using an end-to-end VTB approach that allows us to investigate different types of failures and their responses as seen by the ensemble of PMUs. The collected data on certain types of events may be amenable to certain types of analysis (e.g., alerting for sudden changes can be done on a small window of data) and hence determine the data analytics architectures is required to evaluate the streaming PMU data.

and data analytics architecture.↗

Flow-field characteristics and normal-force correlations for delta wings from Mach 2.4 to 4.6

An experimental investigation has been conducted to determine the upper-surface flowfield types and the normal-force characteristics of a series of delta wing models at supersonic speeds. Flow-visualization data were used to classify the flowfields into seven primary types: shockless attached flow, separation bubble, classical vortex, vortex with shock, shock with no separation, shock-induced separation, and separation bubble with shock. The pressure distributions were integrated to obtain upper and lower surface normal-force loadings. A minimal effect of sweep was observed on the upper-surface normal force at constant Mach number and a minimal effect of Mach number was noted for the 75 deg delta wing lower-surface normal force. The normal-force coefficients for all test conditions were correlated, and a single empirical equation was formulated from which the normal-force coefficient could be calculated as a function of Mach number, angle of attack, and wing aspect ratio.

Covell, Peter F.↗

Unsteady Navier-Stokes computations on a wing-body configuration in ramp motions

Unsteady Navier-Stokes computations are conducted for transonic flows over a wing-body configuration undergoing prescribed ramp motions. The ramp motion from 0 to 15 deg includes angles of attack where vortex breakdown is observed experimentally. The vortex breakdown is found to be delayed until after the ramp motion ends. The dynamic effects on the loads are also demonstrated. The moment coefficient is found to be sensitive to the effect of the virtual mass of the fluid. To verify the numerical results, a grid refinement study has been performed for both steady and unsteady flow conditions, by using up to approximately one million grid points. The results show that the coarse grid can give integrated quantities reasonably well, whereas the finer grids give a more detailed flow structure. Comparisons are also made with available steady-state experimental data.

Obayashi, Shigeru↗

Shuttle Orbiter Contingency Abort Aerodynamics: Real-Gas Effects and High Angles of Attack

An important element of the Space Shuttle Orbiter safety improvement plan is the improved understanding of its aerodynamic performance so as to minimize the "black zones" in the contingency abort trajectories [1]. These zones are regions in the launch trajectory where it is predicted that, due to vehicle limitations, the Orbiter will be unable to return to the launch site in a two or three engine-out scenario. Reduction of these zones requires accurate knowledge of the aerodynamic forces and moments to better assess the structural capability of the vehicle. An interesting aspect of the contingency abort trajectories is that the Orbiter would need to achieve angles of attack as high as 60deg. Such steep attitudes are much higher than those for a nominal flight trajectory. The Orbiter is currently flight certified only up to an angle of attack of 44deg at high Mach numbers and has never flown at angles of attack larger than this limit. Contingency abort trajectories are generated using the data in the Space Shuttle Operational Aerodynamic Data Book (OADB) [2]. The OADB, a detailed document of the aerodynamic environment of the current Orbiter, is primarily based on wind-tunnel measurements (over a wide Mach number and angle-of-attack range) extrapolated to flight conditions using available theories and correlations, and updated with flight data where available. For nominal flight conditions, i.e., angles of attack of less than 45deg, the fidelity of the OADB is excellent due to the availability of flight data. However, at the off-nominal conditions, such as would be encountered on contingency abort trajectories, the fidelity of the OADB is less certain. The primary aims of a recent collaborative effort (completed in the year 2001) between NASA and Boeing were to determine: 1) accurate distributions of pressure and shear loads on the Orbiter at select points in the contingency abort trajectory space; and 2) integrated aerodynamic forces and moments for the entire vehicle and the control surfaces (body flap, speed brake, and elevons). The latter served the useful purpose of verification of the aerodynamic characteristics that went into the generation of the abort trajectories.

Prabhu, Dinesh K.↗

Automated Adversary-in-the-Loop Cyber-Physical Defense Planning

Security of cyber-physical systems (CPS) continues to pose new challenges due to the tight integration and operational complexity of the cyber and physical components. To address these challenges, this article presents a domain-aware, optimization-based approach to determine an effective defense strategy for CPS in an automated fashion—by emulating a strategic adversary in the loop that exploits system vulnerabilities, interconnection of the CPS, and the dynamics of the physical components. Our approach builds on an adversarial decision-making model based on a Markov Decision Process (MDP) that determines the optimal cyber (discrete) and physical (continuous) attack actions over a CPS attack graph. The defense planning problem is modeled as a non-zero-sum game between the adversary and defender. We use a model-free reinforcement learning method to solve the adversary’s problem as a function of the defense strategy. We then employ Bayesian optimization (BO) to find an approximate best-response for the defender to harden the network against the resulting adversary policy. This process is iterated multiple times to improve the strategy for both players. We demonstrate the effectiveness of our approach on a ransomware-inspired graph with a smart building system as the physical process. Numerical studies show that our method converges to a Nash equilibrium for various defender-specific costs of network hardening.

97 MATHEMATICS AND COMPUTING↗

Structures of ATP-bound DNA ligase D in a closed domain conformation reveal a network of amino acid and metal contacts to the ATP phosphates

DNA ligases are the sine qua non of genome integrity and essential for DNA replication and repair in all organisms. DNA ligases join 3'-OH and 5'-PO 4 ends via a series of three nucleotidyl transfer steps. In step 1, ligase reacts with ATP or NAD + to form a covalent ligase-(lysyl-N$ζ$)–AMP intermediate and release pyrophosphate (PP i ) or nicotinamide mononucleotide. In step 2, AMP is transferred from ligase-adenylate to the 5'-PO 4 DNA end to form a DNA-adenylate intermediate (AppDNA). In step 3, ligase catalyzes attack by a DNA 3'-OH on the DNA-adenylate to seal the two ends via a phosphodiester bond and release AMP. Eukaryal, archaeal, and many bacterial and viral DNA ligases are ATP-dependent. The catalytic core of ATP-dependent DNA ligases consists of an N-terminal nucleotidyltransferase domain fused to a C-terminal OB domain. Here we report crystal structures at 1.4–1.8 Å resolution of Mycobacterium tuberculosis LigD, an ATP-dependent DNA ligase dedicated to nonhomologous end joining, in complexes with ATP that highlight large movements of the OB domain (~50 Å), from a closed conformation in the ATP complex to an open conformation in the covalent ligase-AMP intermediate. The LigD·ATP structures revealed a network of amino acid contacts to the ATP phosphates that stabilize the transition state and orient the PP i leaving group. A complex with ATP and magnesium suggested a two-metal mechanism of lysine adenylylation driven by a catalytic Mg 2+ that engages the ATP $α$ phosphate and a second metal that bridges the ATP $β$ and $γ$ phosphates.

59 BASIC BIOLOGICAL SCIENCES↗

Countering Weapons of Mass Destruction (CWMD) Device Cybersecurity Characterization Process and Profile

Countering Weapons of Mass Destruction (CWMD) recognizes that threats in the cyberspace domain continue to grow, which requires CWMD devices and supporting systems to be both cybersecure (ability to protect or defend from cyber-attacks) and resilient (ability to maintain required capability in the face of adversity) to cyber threats. The CWMD cybersecurity characterization approach in this document supports existing cyber resilience activities within the Acquisition Lifecycle Framework. Similarly, this process supports existing Department of Homeland Security Cyber Resilience Test and Evaluation activities, which consist of iterative processes, starting at the initiation of system acquisition and continuing throughout the entire device and system life cycle. Cyber resilience is the ability of an information system to continue to operate while under attack, even if in a degraded or debilitated state, and to rapidly recover operational capabilities for essential functions after a successful attack. The goal of the security characterization task for CWMD is to support the development of a CBRN device-dependent profile that aligns with device network capabilities and maps to recommended security controls to create a characterization security profile impact levels. The impact levels for CWMD devices should be characterized as Low (L), Moderate (M), High (H) to align with the low, moderate, high control baselines. To estimate the impact levels, the device’s security-related attributes are translated into the security objectives: Confidentiality (C), Integrity (I), and Availability (A), known as the CIA triad. The potential impact for each device can be L, M, H, for devices that connect and transmit different types of data and may have different impact levels. National Institute of Standards and Technology Federal Information Processing Standards Publication 199 states, “the potential impact values assigned to the respective security objectives shall be the highest value from among those security categories that have been determined for each type of information resident on the information system.” As CWMD is determining the cybersecurity impact levels of CBRN devices based on network connections and data transfers, the impact levels are aligned with the associated attributes of network connections and communications. For example, if the device system is connected to a wireless network and transmits different data types based on the confidentiality of the data, the highest impact value for each security objective should represent the device’s CIA impact level. This document is intended to be used by test managers, test team, and program managers.

45 MILITARY TECHNOLOGY, WEAPONRY, AND NATIONAL DEF↗

The Iodine Satellite (iSat) Propellant Feed System - Design and Demonstration

CUBESATS are relatively new spacecraft platforms that are typically deployed from a launch vehicle as a secondary payload, providing low-cost access to space for a wide range of end-users. These satellites are comprised of building blocks having dimensions of 10x10x10 cm3 and a mass of 1.33 kg (a 1-U size). While providing low-cost access to space, a major operational limitation is the lack of a propulsion system that can fit within a CubeSat and is capable of executing high Delta V maneuvers. This makes it difficult to use CubeSats on missions requiring certain types of maneuvers (i.e. formation flying, spacecraft rendezvous). Work has been performed investigating the use of iodine as a propellant for Hall-effect thrusters (HETs) that could subsequently be used to provide a high specific impulse path to CubeSat propulsion. One of the systems under development to support such a technology is the propellant feed system, which must be capable of storing solid iodine propellant, applying heat to sublime the stored solid into the vapor phase, and then control the flow of low-pressure gaseous iodine to both the thruster and cathode. In a test conducted in 2016, a first-generation iodine propellant feed system was integrated with a cathode and Hall thruster. While this test had to be terminated, the feed system in this first test was able to support both cathode and integrated cathode and thruster operation prior to the termination of the test. In the present paper, we describe work performed since that initial integrated test. The effort uses lessons learned from the previous integrated test, retiring risk associated with the iodine propellant feed system, answering open design-space questions, and demonstrating iodine flow control in an integrated system. The work is undertaken at both the component level and then at the integrated subsystem level to systematically improve the feed system design, improving the hardware fidelity so the appearance and operation of the system are as flight-like as possible. At the component level, the work focuses on the propellant tank, the feed system tubing, the valves used to control the flow to the cathode and thruster, and the heaters that maintain the temperature of the flowpaths and keep iodine from redepositing and clogging the system. Work on the propellant reservoir focuses on fabricating a tank that matches the geometry of the flight design, which allows for the identification of flight tank fabrication issues that may arise and permits thermal testing of a tank possessing the same size and thermal mass as the flight design, which can be used to anchor thermal modeling of the component. This is critical for finalizing the tank heater power requirements that feed into the heater design. All metallic materials in the feed system are hastelloy or Inconel, as these materials are resistant to chemical attack by the highly-reactive iodine vapor. The tubing in the iodine feed system must possess ports to permit a neutral gas purge of the system that clear impurities after iodine is loaded into the propellant tank. A procedure is discussed whereby these ports are crimped and sealed after the purge process is completed so as to not re-expose the iodine system to air. The valves are a critical component for control of the flow to the thruster and the cathode. Significant effort has gone into upgrading the materials of the valves to make them more resistant to chemical attack and into developing an understanding of the use of these valves during the startup and operation of the cathode and thruster. The heaters that line the entire feed system are designed to draw minimal power from the power processing unit (PPU) while still having the capacity to maintain all the feed system components at the temperatures required to discourage iodine deposition inside components downstream of the propellant tank exit. The heaters possess two separate resistive traces, giving the design redundancy should a failure occur in the primary heater circuit of one of the heater zones. The task of operating a feed system in conjunction with a thruster and cathode is undertaken in a series of sub-steps. The system is first assembled and operated on xenon gas, using the valves for cathode startup and thruster control based on measurement of the discharge current. After startup and control on xenon are demonstrated, the thruster will be transitioned to iodine operation, demonstrating thruster startup and feed system control while using a xenon-fed cathode. Finally, the last step is to integrate an iodine-compatible cathode with the system, demonstrate autonomous cathode start-up with open-loop control and thruster start-up with closed-loop control for multiple cycles.

Polzin, Kurt A.↗

Optimal Client Sampling in Federated Learning with Client-level Heterogeneous Differential Privacy

Federated Learning with client-level differential privacy (DP) provides a promising framework for collaboratively training models while rigorously protecting clients’ privacy. However, classic approaches like DP-FedAvg struggle when clients have heterogeneous privacy requirements, as they must uniformly enforce the strictest privacy level across all clients, leading to excessive DP noise and significant degradation in model utility. Existing methods to improve the model utility in such heterogeneous privacy settings often assume a trusted server and are largely heuristic, resulting in suboptimal performance and lacking strong theoretical foundations. Here, in this work, we address these challenges under a practical attack model where both clients and the server are honest-but-curious. We propose GDPFed, which partitions clients into groups based on their privacy budgets and achieves client-level DP within each group to reduce the privacy budget waste and hence improve the model utility. Based on the privacy and convergence analysis of GDPFed, we find that the magnitude of DP noise depends on both model dimensionality and the per-group client sampling ratios. To further improve the performance of GDPFed, we introduce GDPFed+, which integrates model sparsification to eliminate unnecessary noise and optimizes per-group client sampling ratios to minimize convergence error. Extensive empirical evaluations on multiple benchmark datasets demonstrate the effectiveness of GDPFed+, showing substantial performance gains compared with state-of-the-art methods.

Xu, Jiahao [Univ. of Nevada, Reno, NV (United Stat↗

The role of nutrients in drought‐induced tree mortality and recovery

Summary Global forests are experiencing rising temperatures and more severe droughts, with consistently dire forecasts for negative future impacts. Current research on the physiological mechanisms underlying drought impacts is focused on the water‐ and carbon‐associated mechanisms. The role of nutrients is notably missing from this research agenda. Here, we investigate what role, if any, forest nutrition plays for survival and recovery of forests during and after drought. High nutrient availability may play a detrimental role in drought survival due to preferential biomass allocation aboveground that (1) predispose plants to hydraulic constraints limiting photosynthesis and promoting hydraulic failure, (2) increases carbon costs during periods of carbon starvation, and (3) promote biotic attack due to low tissue carbon: nitrogen (C : N). When nutrient uptake occurs during drought, high nutrient availability can increase water use efficiency thus minimizing negative feedbacks between carbon and nutrient balance. Nutrients are released after drought ceases, which might promote faster recovery but the temporal dynamics of microbial immobilization and nutrient leaching have a significant impact on nutrient availability. We provide a framework for understanding nutrient impacts on drought survival that allows a more complete analysis of forest ecosystem responses. Contents Summary 513 I. Introduction 513 II. Integrating nutrients into the hydraulic framework predicting mechanisms of drought survival, mortality, and recovery 514 III. Integration 517 Acknowledgements 518 References 518

Gessler, Arthur↗

Nacelle Aerodynamic and Inertial Loads (NAIL) project

Inlet airloads were measured by integration of pressures recorded at 252 locations on the right hand inboard (JT9D-7A) nacelle of the Boeing 747 aircraft. Pressures were recorded at 45 locations on the right hand outboard nacelle for comparison. Inertial loads were measured on both nacelles using accelerometers and rate gyros. Flight conditions included takeoffs at several gross weights, high g turns, and a simulated acceptance flight. Airloads at takeoff rotation were found to be larger than at any other normal service condition because of the combined effects of high angle of attack and high engine airflow. Inertial loads were smaller than previous estimates had indicated. A procedure is given for estimating inlet airloads at low speeds and high angles of attack for any underwing high bypass ratio turbofan installation approximately resembling the one tested. Flight procedure modifications are suggested that may result in better fuel economy retention in service. Pressures were recorded on the core cowls and pylons of both engine installations and on adjacent wing surfaces for use in development of computer codes for analysis of installed propulsion system aerodynamic drag interference effects.

Source record↗

Effect of location of aft-mounted nacelles on the longitudinal aerodynamic characteristics of a high-wing transport airplane

As part of a propulsion/airframe integration program at Langley Research Center, tests were conducted in the Langley 16-Foot Transonic Tunnel to determine the effects of locating flow-through mixed flow engine nacelles in several aft underwing positions on the longitudinal aerodynamics of a high wing transport airplane. D-shaped inlet nacelles were used in the test. Some configurations with antishock bodies and with nacelle toe-in were also tested. Data were obtained for a free stream Mach number range of 0.70 to 0.85 and a model angle-of-attack range from -2.5 to 4.0 degrees.

Abeyounis, William K.↗

Assessment of Noise Reduction Concepts for Leading-Edge Slat Noise

The leading-edge slat of a high-lift airfoil can be a significant noise contributor during aircraft landing. This paper summarizes the effects of several passive noise reduction devices on the 30P30N high-lift airfoil. Experiments are conducted on a two-dimensional multi-element high-lift airfoil with leading-edge slat extensions, gap filler, and cove filler in an anechoic wind tunnel to evaluate the effect of passive flow control on the acoustics generated by the unsteady flow field. Slat geometry modifications associated with the treatments alter the flow field in the region that dominates the generation of the acoustic field. Three angles of attack (a(k) = 8°, 10°, and 15.5°) and three different Reynolds numbers (Re(c) = 1.2e6, 1.5e6, and 1.71e6) are selected as the test conditions. Steady surface pressure measurements are conducted to assess the effect of the treatments on the lift and drag. Unsteady surface pressure measurements along with the far-field acoustic array measurements are performed to evaluate the changes in near- and far-field pressure fluctuations, respectively. Delay and Sum (DAS) beamforming method is applied to locate the noise sources on the model and provide integrated spectra. Implementation difficulties with the gap filler led to structural integration deficiencies that prevented a fair assessment of this technology. Among the other passive devices, the cove filler s the most effective noise reduction, along with a negligible change in the aerodynamic metrics.

Zhang, Yang↗

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate C $\bar{β}$ and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗