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DOE OSTI2025

Structural and functional analyses of SARS-CoV-2 Nsp3 and its specific interactions with the 5’ UTR of the viral genome

ABSTRACT Non-structural protein 3 (Nsp3) is the largest open reading frame encoded in the SARS-CoV-2 genome, essential for the formation of double-membrane vesicles (DMV) wherein viral RNA replication occurs. We conducted an extensive structure-function analysis of Nsp3 and determined the crystal structures of the ubiquitin-like 1 (Ubl1), nucleic acid binding (NAB), β-coronavirus-specific marker (βSM) domains, and a sub-region of the Y domain of this protein. We show that the Ubl1, ADP-ribose phosphatase (ADRP), human SARS Unique (HSUD), NAB, and Y domains of Nsp3 bind the 5’ UTR of the viral genome and that the Ubl1 and Y domains possess affinity for recognition of this region, suggesting high specificity. The Ubl1-Nucleocapsid (N) protein complex binds the 5’ UTR with greater affinity than the individual proteins alone. Our results suggest that multiple domains of Nsp3, particularly Ubl1 and Y, shepherd the 5’ UTR of the viral genome during translocation through the DMV membrane, priming the Ubl1 domain to load the genome onto N protein. IMPORTANCE The largest protein encoded by the SARS-CoV-2 genome is Nsp3. In infected cells, this multi-domain protein forms a pore structure in the virus-induced double-membrane vesicles (DMV). We have incomplete data on Nsp3 molecular structure, and here, we describe crystal structures for multiple domains of Nsp3. It is thought that newly replicated viral RNA transits through the DMV pore; however, we possess incomplete data on which regions of Nsp3 actually interact with RNA. Here, we present data showing that five domains of Nsp3 interact with the 5’ UTR of the SARS-CoV-2 RNA, including the Y domain for which no function has ever been discovered. These data suggest that the pore structure plays an active role in recognizing the terminal end of the genome, transiting and loading the viral RNA onto the cytoplasmic nucleocapsid protein. These data help expand our knowledge of Nsp3 structure and function and the SARS-CoV-2 replication cycle.

Microbiology↗

DOE OSTI2021

From Gargamelle to MINERvA: exploring the structure of the nucleon with neutrinos

Neutrino scattering experiments have been exploring the structure of the nucleon with Deep-Inelastic Scattering for over 50 years. Although hints of nucleon structure were available using CERN’s first neutrino beam in the 1960s, the study actually started quantitatively in the early 1970s with the Gargamelle heavy liquid bubble chamber that produced first neutrino confirmation of scaling. This study of nucleon structure continued with both bubble chambers and more massive electronic detectors that, with higher energy neutrino beams and $Q^2$ reach, examined the breaking of this scaling while testing Quantum Chromodynamics. Here, a significant factor when including neutrino explorations of nucleon structure in the overall picture is that to gather significant statistics, neutrino experiments have had to use heavier nuclear targets. It has been experimentally demonstrated that the structure of the nucleon in the nuclear environment is indeed different than the free nucleon structure. Understanding this modified structure has played an important part in the experimental and theoretical exploration of nucleon structure by neutrinos and is one of the important goals of the on-going MINERvA experiment at Fermilab.

72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS↗

DOE OSTI2025

Confocal Raman Microscopy for Measuring In Situ Temperature-Dependent Structural Changes in Poly(Ethylene Oxide) Thin Films

Crystallization from the melt is a critical process governing the properties of semi-crystalline polymeric materials. While structural analyses of melting and crystallization transitions in bulk polymers have been widely reported, in contrast, those in thin polymer films on solid supports have been underexplored. Herein, in situ Raman microscopy and self-modeling curve resolution (SMCR) analysis are applied to investigate the temperature-dependent structural changes in poly(ethylene oxide) (PEO) films during melting and crystallization phase transitions. By resolving complex overlapping sets of spectra, SMCR analysis reveals that the thermal transitions of 50 µm thick PEO films comprise two structural phases: an ordered crystalline phase and a disordered amorphous phase. The ordered structure of the crystalline PEO film entirely disappears as the polymer is heated; conversely, the disordered structure of the amorphous PEO film reverts to the ordered structure as the polymer is cooled. Broadening of the Raman bands was observed in PEO films above the melting temperature (67 °C), while sharpening of bands was observed below the crystallization temperature (45 °C). The temperatures at which these spectral changes occurred were in good agreement with differential scanning calorimetry (DSC) measurements, especially during the melting transition. The results illustrate that in situ Raman microscopy coupled with SMCR analysis is a powerful approach for unraveling complex structural changes in thin polymer films during melting and crystallization processes. Furthermore, we show that confocal Raman microscopy opens opportunities to apply the methodology to interrogate the structural features of PEO or other surface-supported polymer films as thin as 2 µm, a thickness regime beyond the reach of conventional thermal analysis techniques.

Koh, Miharu [Department of Chemistry, University o↗

DOE OSTI2020

The crystal structure of a Polerovirus exoribonuclease-resistant RNA shows how diverse sequences are integrated into a conserved fold

Exoribonuclease-resistant RNAs (xrRNAs) are discrete elements that block the progression of 5' to 3' exoribonucleases using specifically folded RNA structures. A recently discovered class of xrRNA is widespread in several genera of plant-infecting viruses, within both noncoding and protein-coding subgenomic RNAs. The structure of one such xrRNA from a dianthovirus revealed three-dimensional details of the resistant fold but did not answer all questions regarding the conservation and diversity of this xrRNA class. Here, we present the crystal structure of a representative polerovirus xrRNA that contains sequence elements that diverge from the previously solved structure. This new structure rationalizes previously unexplained sequence conservation patterns and shows interactions not present in the first structure. Together, the structures of these xrRNAs from dianthovirus and polerovirus genera support the idea that these plant virus xrRNAs fold through a defined pathway that includes a programmed intermediate conformation. This work deepens our knowledge of the structure-function relationship of xrRNAs and shows how evolution can craft similar RNA folds from divergent sequences.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Structure of the dengue virus RNA promoter

Dengue virus, a single-stranded positive sense RNA virus, is the most prevalent mosquito-borne pathogen in the world. Like all RNA viruses, it uses conserved structural elements within its genome to control essential replicative steps. A 70 nt stem–loop RNA structure (called SLA), found at the 5'-end of the genome of all flaviviruses, functions as the promoter for viral replication. This highly conserved structure interacts with the viral polymerase NS5 to initiate RNA synthesis. Here, we report the NMR structure of a monomeric SLA from dengue virus serotype 1, assembled to high-resolution from independently folded structural elements. The DENV1 SLA has an L-shaped structure, where the top and side helices are coaxially stacked, and the bottom helix is roughly perpendicular to them. Because the sequence is highly conserved among different flavivirus genomes, it is very likely that the three-dimensional fold and local structure of SLA are also conserved among flaviviruses and required for efficient replication. This work provides structural insight into the dengue promoter and provides the foundation for the discovery of new antiviral drugs that target this essential replicative step.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Assessment of Grizzly Capabilities for Reactor Pressure Vessels and Reinforced Concrete Structures

Over the last several years, capabilities to simulate the progression and effects of degradation in critical structures in light water reactor (LWR) nuclear power plants have been under development in the Grizzly code. Age-related material degradation is important for a number of systems in LWRs, but the main focus for Grizzly development has been on reactor pressure vessels (RPVs) and concrete structures because of their central role and the difficulty of replacement of these structures if they are found to be degraded to an unacceptable degree. The capabilities for analyzing both RPVs and concrete structures in Grizzly have reached a point where the feature sets are sufficiently complete to perform credible analyses of those types of structures. Because of this, the emphasis has shifted from foundational development to assessing the accuracy of these modeling capabilities on representative problems of interest and on improving the physical basis of those models to improve their ability to predict the actual response of those structural systems. This report documents a first set of test cases that have been developed to assess the Grizzly code on real-world problems for these two types of structures. For RPVs, these test cases consist of a set of benchmark problems where Grizzly is compared to another code. For concrete structures, a set of models of experimental specimens designed to characterize the multidimensional swelling response of reinforced concrete members due to alkali-silica reaction (ASR) has been developed and compared with experimental results. Both the RPV and concrete test cases developed here are much more computationally intensive than the small regression tests that are used in the testing that Grizzly undergoes every time a proposed set of changes is made to ensure that those changes do not adversely affect previously established behavior. A system for regularly running these large problems to monitor the behavior of the code as it is developed has been instituted. While these test cases generally indicate good comparison with the benchmark results, they also indicate areas where further development is warranted.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗

DOE OSTI2020

Ambient Ion Trapping and Separations for High Throughput Structurally Selective Material Deposition

While ion mobility coupled to mass spectrometry (IMS/MS) has been increasingly adapted to biological analysis, the structural and molecular specificity offered by IMS/MS has untapped potential for creating novel materials (for energy, catalysis, biomolecular switches etc.) with rationally tailored properties by precisely controlling the chemical structure and composition of these materials. While electrochemical and catalytic properties of the bulk material are understood by mass-selective ion-soft-landing, different structures of one molecule can exhibit distinct properties from the bulk. Many biological phenomenon occur at interfaces and are highly structurally specific. IMS/MS offers means to separate molecules in a sample (which is first ionized and released to gas phase from the condensed phase) based on mass and structure. Species thus separated can potentially be deposited onto surfaces with molecular and structural specificity, to perform fundamental investigations for understanding critical interfacial phenomenon. Key performance metrics for IMS/MS analysis are resolution of separations and sensitivity. Recently developed Structures for Lossless Ion Manipulations (SLIM)4 enabled unprecedented resolution in IMS separations. In addition, high sensitivity was enabled in SLIM due to lossless confinement of ions using radio frequency fields. Thus, SLIM enabled extremely long path length separations (up to 0.1 km thus far) providing efficient separation of molecular ions never before achieved (e.g. isotopologues, D&L amino acids). While SLIM has unprecedented analytical separations power/utility, the use of SLIM platform for depositing mobility and mass selected molecular ions has two specific technological challenges: (a) Pulsed nature of IMS separations and their low duty cycle and (b) the need for a low vacuum of ~4 torr for lossless ion confinement. These limitations lead to long deposition times due to loss of ions generated from the sample. Enabling ion confinement at atmospheric pressure will be path-breaking contribution to the fundamental understanding of ion manipulations and separations and will also enable novel science currently precluded due to the inability of the present technologies to confine ions under ambient conditions. Traditionally used radio frequency fields are ineffective in confining ions at high pressures due to ion/neutral collisions. To address these, we propose to (a) develop methods for confining and manipulating ions for lossless IMS separations at atmospheric pressure enabling high sensitivity and throughput and (b) use the high specificity of IMS/MS and high throughput from ambient ion confinement to perform mobility and mass selective ion deposition at atmospheric pressure. Our goal is to enable the study of the fundamental properties of energy/catalysis materials and biomolecules. This project paves the way for high-throughput, mass and structure selective material deposition for highly specific surface creation/functionalization (discussed in Section 2 of the report). Further, the developments reported continue to push the boundaries of this process to ambient conditions (discussed in Section 3 of the report). We conclude with a list of specific intellectual property and publications which have been the outcome of this funded project.

36 MATERIALS SCIENCE↗

DOE OSTI2026

Doping Metallic Grain Boundaries to Control Atomic Structure and Damage Tolerance

Grain boundaries often act as sites for crack and void nucleation during plastic deformation of metallic materials. While it is known that grain boundary character and structure can greatly influence this damage nucleation process, the current level of control over such details is limited. The objective of this project was to obtain a fundamental understanding of how metallic grain boundary structure can be controlled through intelligent doping, with the idea of inducing planned amorphous grain boundary phases or complexions. The effect of amorphous complexion structure on dislocation accommodation mechanisms was studied, to improve the field’s understanding of damage nucleation at a promising type of interface. Different microstructural descriptors were studied, as grain boundaries can have large variations and complexity of local structure, and influence the mechanical damage resistance of these features was tested. Nanocrystalline systems that contain amorphous grain boundary complexions were prioritized, as these have more extreme variations in interfacial structure and damage tolerance, with an emphasis on isolating the importance of complexion population (type, thickness, etc.), network topology, local structure, and local chemistry. This research used a combination of computational, experimental, and characterization techniques to isolate and understand the importance of nanoscale grain boundary structure and interfacial chemistry. Amorphous grain boundary complexions were found to clearly increase a material’s resistance to mechanical damage, with the local distribution of structural short-range order within the complexions found to be an important descriptor for damage and the proposed focus of future work in this area.

36 MATERIALS SCIENCE↗

DOE OSTI2020

The significant impact of aerosol vertical structure on lower atmosphere stability and its critical role in aerosol–planetary boundary layer (PBL) interactions

The aerosol–planetary boundary layer (PBL) interaction was proposed as an important mechanism to stabilize the atmosphere and exacerbate surface air pollution. Despite the tremendous progress made in understanding this process, its magnitude and significance still have large uncertainties and vary largely with aerosol distribution and meteorological conditions. In this study, we focus on the role of aerosol vertical distribution in thermodynamic stability and PBL development by jointly using micropulse lidar, sun photometer, and radiosonde measurements taken in Beijing. Despite the complexity of aerosol vertical distributions, cloud-free aerosol structures can be largely classified into three types: well-mixed, decreasing with height, and inverse structures. The aerosol–PBL relationship and diurnal cycles of the PBL height and PM 2.5 associated with these different aerosol vertical structures show distinct characteristics. The vertical distribution of aerosol radiative forcing differs drastically among the three types, with strong heating in the lower, middle, and upper PBL, respectively. Such a discrepancy in the heating rate affects the atmospheric buoyancy and stability differently in the three distinct aerosol structures. Absorbing aerosols have a weaker effect of stabilizing the lower atmosphere under the decreasing structure than under the inverse structure. As a result, the aerosol–PBL interaction can be strengthened by the inverse aerosol structure and can be potentially neutralized by the decreasing structure. Moreover, aerosols can both enhance and suppress PBL stability, leading to both positive and negative feedback loops. This study attempts to improve our understanding of the aerosol–PBL interaction, showing the importance of the observational constraint of aerosol vertical distribution for simulating this interaction and consequent feedbacks.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS1971

Structural interaction with control systems

A monograph which assesses the state of the art of space vehicle design and development is presented. The monograph presents criteria and recommended practices for determining the structural data and a mathematical structural model of the vehicle needed for accurate prediction of structure and control-system interaction; for design to minimize undesirable interactions between the structure and the control system; and for determining techniques to achieve the maximum desirable interactions and associated structural design benefits. All space vehicles are treated, including launch vehicles, spacecraft, and entry vehicles. Important structural characteristics which affect the structural model used for structural and control-system interaction analysis are given.

Noll, R. B.↗

NASA NTRS1977

Analysis of simple 2-D and 3-D metal structures subjected to fragment impact

Theoretical methods were developed for predicting the large-deflection elastic-plastic transient structural responses of metal containment or deflector (C/D) structures to cope with rotor burst fragment impact attack. For two-dimensional C/D structures both, finite element and finite difference analysis methods were employed to analyze structural response produced by either prescribed transient loads or fragment impact. For the latter category, two time-wise step-by-step analysis procedures were devised to predict the structural responses resulting from a succession of fragment impacts: the collision force method (CFM) which utilizes an approximate prediction of the force applied to the attacked structure during fragment impact, and the collision imparted velocity method (CIVM) in which the impact-induced velocity increment acquired by a region of the impacted structure near the impact point is computed. The merits and limitations of these approaches are discussed. For the analysis of 3-d responses of C/D structures, only the CIVM approach was investigated.

Witmer, E. A.↗

NASA NTRS1977

Study of metallic structural design concepts for an arrow wing supersonic cruise configuration

A structural design study was made, to assess the relative merits of various metallic structural concepts and materials for an advanced supersonic aircraft cruising at Mach 2.7. Preliminary studies were made to ensure compliance of the configuration with general design criteria, integrate the propulsion system with the airframe, select structural concepts and materials, and define an efficient structural arrangement. An advanced computerized structural design system was used, in conjunction with a relatively large, complex finite element model, for detailed analysis and sizing of structural members to satisfy strength and flutter criteria. A baseline aircraft design was developed for assessment of current technology. Criteria, analysis methods, and results are presented. The effect on design methods of using the computerized structural design system was appraised, and recommendations are presented concerning further development of design tools, development of materials and structural concepts, and research on basic technology.

Turner, M. J.↗

NASA NTRS1979

Weight optimization of ultra large space structures

The paper describes the optimization of a solar power satellite structure for minimum mass and system cost. The solar power satellite is an ultra large low frequency and lightly damped space structure; derivation of its structural design requirements required accommodation of gravity gradient torques which impose primary loads, life up to 100 years in the rigorous geosynchronous orbit radiation environment, and prevention of continuous wave motion in a solar array blanket suspended from a huge, lightly damped structure subject to periodic excitations. The satellite structural design required a parametric study of structural configurations and consideration of the fabrication and assembly techniques, which resulted in a final structure which met all requirements at a structural mass fraction of 10%.

Reinert, R. P.↗

NASA NTRS1983

Structures and subsystems

New and improved materials combined with efficient structural design concepts have made an essential contribution towards the shaping of the present transportation system (STS), and now, for the replacement of the STS in the year 2005, new materials and novel designs are being studied to identify the technologies which should be developed for a low-cost future space transportation system (FSTS). Three basic structural arrangements were considered for the FSTS orbiter. They include a nonintegral tank arrangement, an integral-tank arrangement, and a hybrid of the first two. Three representative arrangements regarding wall constructions are considered. Each employs a blade-stiffened aluminum tank with reinforced closed-cell-foam cryogenic insulation. Attention is given to an aluminum-alloy structure, a graphite-epoxy structure, a graphite-polyimide structure, a carbon-carbon surface panel structure, a graphite-composite fuselage structure, serviceability and all-weather considerations, and structural concept ratings.

Taylor, A. H.↗

NASA NTRS1985

Computational structural mechanics: A new activity at the NASA Langley Research Center

Complex structures considered for the late 1980's and early 1990's include composite primary aircraft structures and the space station. These structures are much more difficult to analyze than today's structures and necessitate a major upgrade in computerized structural analysis technology. A major research activity in computational structural mechanics (CSM) was initiated. The objective of the CSM activity is develop advanced structural analysis technology that will exploit modern and emerging computers such as computers with vector and/or parallel processing capabilities. The three main research activities underway in CSM include: (1) structural analysis methods development; (2) a software testbed for evaluating the methods; and (3) numerical techniques for parallel processing computers. The motivation and objectives of the CSM activity are presented and CSM activity is described. The current CSM research thrusts, and near and long term CSM research thrusts are outlined.

Knight, N. F., Jr.↗

NASA NTRS1986

Propeller-induced structure-borne noise - Laboratory-based test apparatus

A potentially important source of structure-borne interior noise transmission in advanced turboprop aircraft is the impingement of the propeller wake/vortex on downstream aerodynamic surfaces. The expected levels of propeller wake/vortex-induced structure-borne noise transmission are not known nor can they be determined with present-day technology. A test apparatus has been designed, built and calibrated for the purposes of studying propeller-induced, structure-borne noise transmission in prototypical aircraft structures. The principal approach to the test apparatus design was to provide a physical means of separating the airborne and structure-borne noise components so that the structure-borne noise transmission response could be studied directly without airborne noise contamination. This was accomplished by housing the receiving fuselage structure in an acoustic shield fitted with a wing-to-fuselage acoustic seal. Initial evaluation of the wing-to-fuselage acoustic seal indicates adequate airborne noise isolation to allow direct study of structure-borne noise transmission.

Unruh, J. F.↗

NASA NTRS1986

Design considerations for joints in deployable space truss structures

All of the structures considered for the Control of Flexible Structures (COFS) flight experiments are deployable truss structures and their response will be dominated by the structural response of the joints. To prepare for these experiments some fundamental research work is being conducted in the Structures and Dynamics Division at LaRC which will provide insight into structurally efficient and predictable deployable truss joints. This work involves generic studies of the static and dynamic response of joints as well as the development of analytical models which can be used to predict the response of a large multijointed truss. In addition to the generic joint studies, the research effort encompasses the design and fabrication of a 20-meter long deployable truss beam for laboratory evaluation of its structural characteristics and correlation with developed prediction methods. The experimental results have indicated the importance of attention to detail in the design and fabrication of joints for deployable truss structures. The dimensional relations and material considerations for efficient pin-clevis joints have been outlined. Results of tests on the near-center latch are discussed.

Rhodes, Marvin D.↗

NASA NTRS1987

Optimization procedure to control the coupling of vibration modes in flexible space structures

As spacecraft structural concepts increase in size and flexibility, the vibration frequencies become more closely-spaced. The identification and control of such closely-spaced frequencies present a significant challenge. To validate system identification and control methods prior to actual flight, simpler space structures will be flown. To challenge the above technologies, it will be necessary to design these structures with closely-spaced or coupled vibration modes. Thus, there exists a need to develop a systematic method to design a structure which has closely-spaced vibration frequencies. This paper describes an optimization procedure which is used to design a large flexible structure to have closely-spaced vibration frequencies. The procedure uses a general-purpose finite element analysis program for the vibration and sensitivity analyses and a general-purpose optimization program. Results are presented from two studies. The first study uses a detailed model of a large flexible structure to design a structure with one pair of closely-spaced frequencies. The second study uses a simple equivalent beam model of a large flexible structure to obtain a design with two pairs of closely-spaced frequencies.

Walsh, Joanne L.↗