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At least 217 records · Page 12

Structural analysis of the NifL-NifA complex reveals the molecular basis of anti-activation of nitrogen fixation gene expression in Azotobacter vinelandii

Understanding the molecular basis of regulated nitrogen (N 2 ) fixation is essential for engineering N 2 -fixing bacteria that fulfill the demand of crop plants for fixed nitrogen, reducing our reliance on synthetic nitrogen fertilizers. In Azotobacter vinelandii and many other members of Proteobacteria, the two-component system comprising the anti-activator protein (NifL) and the Nif-specific transcriptional activator (NifA)controls the expression of nif genes, encoding the nitrogen fixation machinery. The NifL-NifA system evolved the ability to integrate several environmental cues, such as oxygen, nitrogen, and carbon availability. The nitrogen fixation machinery is thereby only activated under strictly favorable conditions, enabling diazotrophs to thrive in competitive environments. While genetic and biochemical studies have enlightened our understanding of how NifL represses NifA, the molecular basis of NifA sequestration by NifL depends on structural information on their interaction. Here, we present mechanistic insights into how nitrogen fixation is regulated by combining biochemical and genetic approaches with a low-resolution cryo-electron microscopy (cryo-EM) map of the oxidized NifL-NifA complex. Our findings define the interaction surface between NifL and NifA and reveal how this interaction can be manipulated to generate bacterial strains with increased nitrogen fixation rates able to secrete surplus nitrogen outside the cell, a crucial step in engineering improved nitrogen delivery to crop plants.

59 BASIC BIOLOGICAL SCIENCES↗

The Grain Boundary Relaxation (GBR) Approach for Manufacturing High Strength Nanocrystalline Lightweight Metals

The overarching goal of the project was to conduct research and development work as proposed in the Statement of Project Objective (SOPO) of the award document DE-FE-0009116. The project had 4 tasks and 12 milestones. All the milestone deliverables were completed. The accomplishments of the project objectives and technical discussions are described in Sections 3 and 4, respectively. The modeling and simulation work indicated that to increase the strength and stability of nanocrystalline aluminum (Al), selection of dopants, such as Mg, is necessary. It was predicted that the crystallite size should be less than 50 nm to give high strength. On the basis of modeling, cryo-milling of Al was conducted with the addition of Mg as a function of different times. The crystallite size of the cryo-milled powders was determined by XRD and TEM. Both measurements showed that the actual crystallite size of the grain was <40 nm. The thermal stability of the grain size was established as a function of temperature. It was established that the grain size was < 50 nm up to 500C. The crystallite size of the bulk sample prepared by spark plasma sintering (SPS) and cold spray (CS) additive manufacturing was less than <40 nm. The mechanical properties of the bulk samples prepared by SPS and CS, showed excellent microhardness, good tensile properties (>200 MPa) with moderate ductility and improved fatigue performance. Adding yttria stabilized zirconia (YSZ) improved the build thick of the CS sample, however the YSZ was getting embedded into the sample. A highly dense SPS samples sent for 3rd party testing to the Innovation Testing Services showed a minimum hardness of 180 HV with an average tensile strength of 512.5 MPa. The high cycle fatigue tests also showed an endurance limit of 179.5 MPa. The Energy cost evaluations showed an overall energy cost of around $\$$17.05 for the cryomilling and SPS processes and the total manufacturing cost calculations of $\$$78.14 for 1 kg of sample. The energy cost to prepare a Kg of CS sample is $\$$17.60 and the overall manufacturing cost is $\$$86.85.

36 MATERIALS SCIENCE↗

De novo synthesis and near atomic resolution imaging of host immune receptors critical for pathogen recognition (Abbreviated Final Report)

The innate immune system serves as the body’s first line of defense against invading pathogens, responding rapidly through the deployment of immune cells at common sites of infection, such as the skin and airways. These immune-cell sentinels express a range of highly conserved receptors, including Toll-like receptors (TLRs), which recognize and bind to pathogen-derived components. This recognition event triggers intracellular signaling cascades that initiate and coordinate immune responses. Despite their significance, the complete structural characterization of full-length TLRs, including their extracellular domain (ECD), transmembrane domain (TMD), and Toll/interleukin-1 receptor (TIR) domain, remains incomplete. In this study, we examined the expression and isolation of human TLR4 incorporated into nanodiscs using two approaches: a cell-free synthesis system and a cell-based transfection strategy employing Expi293F cells derived from the human embryonic kidney lineage. Our findings demonstrate that NLP-bound human TLR4 produced via the cell-based method yielded functional protein suitable for time-resolved single-particle cryo-electron microscopy (cryo-EM). This advancement enables structural characterization of full-length TLR4, maintaining the integrity of its extracellular domain, transmembrane domain, and Toll/Interleukin-1 receptor (TIR) domain.

59 BASIC BIOLOGICAL SCIENCES↗

Towards libraries of different ultrasmall amorphous silica cage topologies

Cage topology controlled at the nanometer length scale is expected to enable original functionalities, e.g., in catalysis and therapeutics. Cages are fundamental structural motifs of clathrates and their topological duals, Frank-Kasper phases, and are constituents of mesoporous silica frameworks. However, with one exception, they have not been synthesized as discrete particles. By varying reactant ratios of surfactant, oil, and silane, we report the discovery of 5(12)6(2) and 5(12)6(8) amorphous silica polyhedra, each coexisting with the previously identified 5(12) cage, using combined cryo-transmission electron microscopy (cryo-TEM) and single-particle reconstruction (SPR). Notably, the 5(12)6(8) polyhedron represents a topology not previously observed in mesoporous silica frameworks. Control over structural features is demonstrated, and insights into cage formation mechanisms are provided. Structural outcomes are summarized in a ternary morphology diagram alongside prior results, bridging serendipitous discovery and intentional design of silica cages displaying a level of control over silica polymerization rivaling nature.

Jang, Dong June [Department of Materials Science a↗

Quantitative Modeling of High-Energy Electron Scattering in Thick Samples Using Monte Carlo Techniques

Cryo-electron microscopy (cryo-EM) is a powerful tool for imaging biological samples but is typically limited by sample thickness, which is restricted to a few hundred nanometers depending on the electron energy. However, there is a growing need for imaging techniques capable of studying biological samples up to 10 µm in thickness while maintaining nanoscale resolution. This need motivates the use of mega-electron-volt scanning transmission electron microscopy (MeV-STEM), which leverages the high penetration power of MeV electrons to generate high-resolution images of thicker samples. In this study, we employ Monte Carlo simulations to model electron–sample interactions and explore the signal decay of imaging electrons through thick specimens. By incorporating material properties, interaction cross-sections for energy loss, and experimental parameters, we investigate the relationship between the incident and transmitted beam intensities. Key factors such as detector collection angle, convergence semi-angle, and the material properties of samples were analyzed. Our results demonstrate that the relationship between incident and transmitted beam intensities follows the Beer–Lambert law over thicknesses ranging from a few microns to several tens of microns, depending on material composition, electron energy, and collection angles. The linear depth of silicon dioxide reaches 3.9 µm at 3 MeV, about 6 times higher than that at 300 keV. Meanwhile, the linear depth of amorphous ice reaches 17.9 µm at 3 MeV, approximately 11.5 times higher than that at 300 keV. These findings are crucial for advancing the study of thick biological and semiconductor samples using MeV-STEM.

36 MATERIALS SCIENCE↗

Quality assurance and quality control of the $26~\text {m}^2$ SiPM production for the DarkSide-20k dark matter experiment

DarkSide-20k is a novel liquid argon dark matter detector currently under construction at the Laboratori Nazionali del Gran Sasso (LNGS) of the Istituto Nazionale di Fisica Nucleare (INFN) that will push the sensitivity for Weakly Interacting Massive Particle (WIMP) detection into the neutrino fog. The core of the apparatus is a dual-phase Time Projection Chamber (TPC), filled with 50 tonnes of low radioactivity underground argon (UAr) acting as the WIMP target. NUV-HD-cryo Silicon Photomultipliers (SiPM)s designed by Fondazione Bruno Kessler (FBK) (Trento, Italy) were selected as the photon sensors covering two $10.5~\text {m}^2$ Optical Planes, one at each end of the TPC, and a total of $5~\text {m}^2$ photosensitive surface for the liquid argon veto detectors. This paper describes the Quality Assurance and Quality Control (QA/QC) plan and procedures accompanying the production of FBK NUV-HD-cryo SiPM wafers manufactured by LFoundry s.r.l. (Avezzano, AQ, Italy). SiPM characteristics are measured at 77 K at the wafer level with a custom-designed probe station. As of March 2025, 1314 of the 1400 production wafers (94% of the total) for DarkSide-20k were tested. The wafer yield is $93.2\pm 2.5$%, which exceeds the 80% specification defined in the original DarkSide-20k production plan.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

Metallic alloy stability studies

The dimensional stability of candidate cryogenic wind tunnel model materials was investigated. Flat specimens of candidate materials were fabricated and cryo-cycled to assess relative dimensional stability. Existing 2-dimensional airfoil models as well as models in various stages of manufacture were also cryo-cycled. The tests indicate that 18 Ni maraging steel offers the greatest dimensional stability and that PH 13-8 Mo stainless steel is the most stable of the stainless steels. Dimensional stability is influenced primarily by metallurgical transformations (austenitic to martensitic) and manufacturing-induced stresses. These factors can be minimized by utilization of stable alloys, refinement of existing manufacturing techniques, and incorporation of new manufacturing technologies.

Firth, G. C.↗

Testing experience with unheated stain-gage balances in the NTF

A series of cryogenic (cryo) cycles was conducted in the cryo chamber at the National Transonic Facility (NTF) in order to identify the cause of apparent strain shifts in axial force with temperature for the Pathfinder I model and to minimize their effects. The results of the investigation indicated that the major cause of axial force end point shifts and thermal hysteresis loops was the thickness of the Teflon insulation on the instrumentation wires crossing the balance. By reducing the thickness of the insulation and the total number and size of the wires, apparent strain values were achieved for the model with instrumentation wires which were nearly identical to those for the model without wires. Because of the special design features used, the balance output was very accurate and repeatable over the entire NTF temperature range, even with balance thermal gradients as large as 64 F and transient conditions as large as 3 F/minute.

Jacobs, Peter F.↗

MSFC SBMD Cryogenic Test Data Review

The Subscale Beryllium Mirror Demonstrator (SBMD) was developed in support of the Next Generation Space Telescope program. The goal was to produce a 0.5 m class, ultra lightweight (<12 kg/sq m), beryllium mirror that could be operated at 35 K. The mirror, developed by Ball Aerospace, has been cryogenically tested at the Marshall Space Flight Center (MSFC) a total of three times. The first two tests established the cryogenic performance and repeatability of the SBMD. Between the second and third tests, the mirror was cryo-figured. Thus, the third test measured the performance of the mirror with cryo-figuring applied. This paper will describe the test goals, the test instrumentation, and the test results (low & high order figure and radius of curvature) for all three cryogenic tests.

Hadaway, James B.↗

Hot Views on Cold Crystals: The Application of Thermal Imaging in Cryocrystallography

We have used thermal imaging techniques to visualize the cryocooling processes of macromolecular crystals. Cryocooling is a common technique used for structural data collection to reduce radiation damage in intense X-ray beams and decrease the thermal motion of the atoms. From the thermal images it was clear that during cryocooling a cold wave progresses through a crystal starting at the face closest to the origin of the cold stream and ending at the point furthest away. As an extension to this work, we used thermal imaging to study small crystals, held in a cryo-loop, in the presence of vitrified mother liquor. The different infrared transmission and reflectance properties of the crystal in comparison to the mother liquor surrounding it are thought to be the parameter that produces the contrast that makes the crystal visible. An application of this technology may be the determination of the exact location of small crystals in a cryo-loop for automated structural genomics studies. Data from initial tests in support of application development was recorded for lysozyme crystals and for bFGF/dna complex crystals, which were cryocooled and imaged in large loops, both with visible light and with infrared radiation. The crystals were clearly distinguished from the vitrified solution in the infrared spectrum, while in the case of the bFGF/dna complex the illumination had to be carefully manipulated to make the crystal visible in the visible spectrum. These results suggest that the thermal imaging may be more sensitive than visual imaging for automated location of small crystals. However, further work on small crystals robotically mounted at SSRL did not clearly visualize those crystals. The depth of field of the camera proved to be limiting and a different cooling geometry was used, compared to the previous, successful experiments. Analysis to exploit multiple images to improve depth of field and experimental work to understand cooling geometry effects is ongoing. These results will be presented along with advantages and disadvantages of the technique and a discussion of how it might be applied.

Snell, Eddie↗

Hot Stuff? Thermal Imaging Applied to Cryocrystallography

In the past we have used thermal imaging techniques to visualize the cryocooling processes of macromolecular crystals. From these images it was clear that a cold wave progresses through a crystal starting at the face closest to the origin of the cold stream and ending at the point furthest away. During these studies we used large volume crystals, which were clearly distinguished fiom the loop holding them. These large crystals, originally grown for neutron diffiaction studies, were chosen deliberately to enhance the imaging. As an extension to this work, we present used thermal imaging to study small crystals, held in a cryo-loop, in the presence of vitrified mother liquor. The different d a r e d transmission and reflectance properties of the crystal in comparison to the mother liquor surrounding it are thought to be the parameter that produces the contrast that makes the crystal visible. An application of this technology may be the determination of the exact location of small crystals in a cryo-loop. Data fkom initial tests in support of application development was recorded for lysozyme crystals and for bFGF/dna complex crystals, which were cryocooled and imaged in large loops, both with visible light mad with h i k e d rdi&tion. The crystals were clearly distinguished from the vitrified solution in the infiared spectrum, while in the case of the bFGF/dna complex the illumination had to be carefully manipulated to make the crystal visible in the visible spectrum. These results suggest that the thermal imaging may be more sensitive than visual imaging for automated location of small crystals. However, further work on small crystals robotically mounted at SSRL did not clearly visualize those crystals. The depth of field of the camera proved to be limiting and a different cooling geometry was used, compared to the previous, successful experiments. Analysis to exploit multiple images to improve depth of field and experimental work to understand cooling geometry effects is ongoing. These results will be presented along with advantages and disadvantages of the technique and a discussion of how it might be applied.

Snell, E. H.↗

Cryogenic Test Results of Hextek Mirror

A 250 mm diameter lightweight borosilicate mirror has been interferometrically tested from room-temperature down to 30 K at the X-Ray Calibration Facility (XRCF) at Marshall Space Flight Center (MSFC). The minor blank was manufactured by Hextek Corporation using a high-temperature gas fusion process and was then polished at MSFC. It is a sandwich-type mirror consisting of a thin face-sheet (approx.1.5 mm thick), a core structure (20 mm thick, approx.43 mm diameter cells, & 0.5-1.2 mm thick walls), and a thin back-sheet (3 mm thick). The mirror has a 2500 mm spherical radius-of- curvature @/lo). The areal density is 14 kg/sq m. The mirror was tested in the 1 m x 2 m chamber using an Instantaneous Phase Interferometer (PI) from ADE Phase Shift Technologies. The mirror was tested twice. The first test measured the change in surface figure from ambient to 30 K and the repeatability of the change. An attempt was then made by QED Technologies to cryo-figure the mirror using magnetorheological finishing. The second test measured the effectiveness of the cryo- figuring. This paper will describe the test goals, the test instrumentation, and the test results for these cryogenic tests.

Hadaway, James↗

Cryogenic System for Interferometric Measurement of Dimensional Changes at 40 K: Design and Performance

This report describes the facility, experimental methods, characterizations, and uncertainty analysis of the Cryo Distortion Measurement Facility (CDMF) at the Goddard Space Flight Center (GSFC). This facility is designed to measure thermal distortions of structural elements as the temperature is lowered from 320K to below 40 K over multiple cycles, and is capable of unattended running and data logging. The first measurement is to be the change in length and any bending of composite tubes with Invar end-fittings. The CDMF includes a chamber that is efficiently cooled with two cryo-coolers (one single-stage and one two-stage) rather than with liquid cryogens. Five optical ports incorporate sapphire radiation shields - transparent to the interferometer - on each of two shrouds and a fused silica vacuum-port window. The change in length of composite tubes is monitored continuously with displacement-measuring interferometers; and the rotations, bending, and twisting are measured intermittently with theodolites and a surface-figure interferometer. Nickel-coated invar mirrors and attachment mechanisms were developed and qualified by test in the CDMF. The uncertainty in measurement of length change of 0.4 m tubes is currently estimated at 0.9 micrometers.

Blake, Peter↗

Development of a Cryogenic Thermal Distortion Measurement Facility for Testing the James Webb Space Telescope Instrument Support Integration Module 2-D Test Assemblies

The James Webb Space Telescope Instrument Support Integration Module (ISIM) is being designed and developed at the Goddard Space Flight Center. The ISM Thermal Distortion Testing (ITDT) program was started with the primary objective to validate the ISM mechanical design process. The ITDT effort seeks to establish confidence and demonstrate the ability to predict thermal distortion in composite structures at cryogenic temperatures using solid element models. This-program's goal is to better ensure that ISIM meets all the mechanical and structural requirements by using test results to verify or improve structural modeling techniques. The first step to accomplish the ITDT objectives was to design, and then construct solid element models of a series 2-D test assemblies that represent critical building blocks of the ISIM structure. Second, the actual test assemblies consisting of composite tubes and invar end fittings were fabricated and tested for thermal distortion. This paper presents the development of the GSFC Cryo Distortion Measurement Facility (CDMF) to meet the requirements of the ISIM 2-D test. assemblies, and other future ISIM testing needs. The CDMF provides efficient cooling with both a single, and two-stage cryo-cooler. Temperature uniformity of the test assemblies during thermal transients and at steady state is accomplished by using sapphire windows for all of the optical ports on the radiation shields and by using .thermal straps to cool the test assemblies. Numerical thermal models of the test assemblies were used to predict the temperature uniformity of the parts during cooldown and at steady state. Results of these models are compared to actual temperature data from the tests. Temperature sensors with a 0.25K precision were used to insure that test assembly gradients did not exceed 2K lateral, and 4K axially. The thermal distortions of two assemblies were measured during six thermal cycles from 320K to 35K using laser interferometers. The standard deviation for all of the distortion measurements is less than 0.5 microns, which falls within the ISIM requirement of 3 microns.

Miller, Franklin↗

Terrestrial Planet Finder Interferometer 2005

Activities Affected by Budget Adjustment: Formation Acquisition Sensor, Cryo Structures, Cryo Delay Line, Cryocooler, Design Team, System Modeling. Other Activities include: Formation Flying, Cryogenic Operations, Starlight suppression, and System Design and Modeling.

interferometry↗

The Integration and Test Program of the James Webb Space Telescope

With the delivery of its flight scientific instruments and the completion of all telescope optics both scheduled to occur later this year, the James Webb Space Telescope (JWST) will enter into a challenging integration and test (I&T) program. Highlights of that program include cryo-vacuum tests of the Integrated Science Instrument Module (ISIM) at Goddard Space Flight Center, ambient integration of the ISIM and the Optical Telescope Element (OTE) at Goddard, and an end-to-end cryo-vacuum test of the OTE + ISIM system at Johnson Space Center. We review the overall flow of the I&T program, highlighting the key activities and the critical verifications to be performed at each step.

Randy, Kimble↗

Cryogenic Propulsion Stage

The CPS is an in-space cryogenic propulsive stage based largely on state of the practice design for launch vehicle upper stages. However, unlike conventional propulsive stages, it also contains power generation and thermal control systems to limit the loss of liquid hydrogen and oxygen due to boil-off during extended in-space storage. The CPS provides the necessary (Delta)V for rapid transfer of in-space elements to their destinations or staging points (i.e., E-M L1). The CPS is designed around a block upgrade strategy to provide maximum mission/architecture flexibility. Block 1 CPS: Short duration flight times (hours), passive cryo fluid management. Block 2 CPS: Long duration flight times (days/weeks/months), active and passive cryo fluid management.

Jones, David↗

Analysis of Direct Samples of Early Solar System Aqueous Fluids

Over the past three decades we have become increasingly aware of the fundamental importance of water, and aqueous alteration, on primitive solar-system bodies. Some carbonaceous and ordinary chondrites have been altered by interactions with liquid water within the first 10 million years after formation of their parent asteroids. Millimeter to centimeter-sized aggregates of purple halite containing aqueous fluid inclusions were found in the matrix of two freshly-fallen brecciated H chondrite falls, Monahans (1998, hereafter simply "Monahans") (H5) and Zag (H3-6) (Zolensky et al., 1999; Whitby et al., 2000; Bogard et al., 2001) In order to understand origin and evolution of the aqueous fluids inside these inclusions we much measure the actual fluid composition, and also learn the O and H isotopic composition of the water. It has taken a decade for laboratory analytical techniques to catch up to these particular nanomole-sized aqueous samples. We have recently been successful in (1) measuring the isotopic composition of H and O in the water in a few fluid inclusions from the Zag and Monahans halite, (2) mineralogical characterization of the solid mineral phases associated with the aqueous fluids within the halite, and (3) the first minor element analyses of the fluid itself. A Cameca ims-1270 equipped with a cryo-sample-stage of Hokkaido University was specially prepared for the O and H isotopic measurements. The cryo-sample-stage (Techno. I. S. Corp.) was cooled down to c.a. -190 C using liquid nitrogen at which the aqueous fluid in inclusions was frozen. We excavated the salt crystal surfaces to expose the frozen fluids using a 15 keV Cs+ beam and measured negative secondary ions. The secondary ions from deep craters of approximately 10 m in depth emitted stably but the intensities changed gradually during measurement cycles because of shifting states of charge compensation, resulting in rather poor reproducibility of multiple measurements of standard fluid inclusions of +/- 90 0/00(2 sigma) for delta D, and +/- 29 0/00 (2 sigma) for delta O-18. On the other hand, the reproducibility of Delta O-17 is plus or minus 8 /00 (2 sigma ) because the observed variations of isotope ratios follow a mass dependent fractionation law. Variations of delta D of the aqueous fluids range over sog,a 330(90; 2 sigma ) to +1200(90) 0/00 for Monahans and delta 300(96) 0/00 to +90(98)0/00 for Zag. Delta O-17 of aqueous fluids range over delta 16(22) 0/00 to +18(10) 0/00 for Monahans and +3(10) 0/00 to +27(11) 0/00 for Zag. These variations are larger than the reproducibility of standard analyses and suggest that isotope equilibria were under way in the fluids before trapping into halite. The mean values of delta D and Delta O-17 are +290 0/00 and +9 0/00, respectively. The mean values and the variations of these fluids are different from the representative values of ordinary chondrites, verifying our working hypothesis that the fluid inclusion-bearing halites were not indigenous to the H chondrite parent-asteroid but rather represent exogenous material delivered onto the asteroid from a separate cryovolcanically-active body. This initial isotopic work has demonstrated the feasibility of the measurements, but also revealed sample processing and analytical shortcomings that are now being addressed. Examination of solid mineral inclusions within Monahans and Zag halite grains by confocal Raman spectroscopy at the Carnegie Geophysical Laboratory has revealed them to be metal, magnetite, forsteritic olivine (Fo.98), macromolecular carbon (MMC), pyroxenes, feldspar with Raman spectral affinity to anorthoclase and, probably, fine-grained lepidocrocite (FeO(OH)). In addition, one inclusion features aliphatic material with Raman spectral features consistent with a mixture of short-chain aliphatic compounds. We have initiated analyses of the bulk composition of the fluids within the inclusions in Zag and Monahans halites at Virginia Tech by LA ICPMS using angilent 7500ce quadrupole ICPMS and a Lambda Physik GeoLas 193 nm excimer laser ablation system. Preliminary results reveal that the inclusion aqueous fluids contain highly charged cations of Ca, Mg and Fe. The minerals and compounds discovered thus far within Monahans/Zag halites are indicative of an originating body at least partly composed of unequilibrated anhydrous materials (high Fo olivine, pyroxenes, feldspars, possibly the metal) which were subjected to aqueous alteration (the halite parent brine) and containing a light organic component (the short-chain aliphatic compounds). This material was ejected from the originating body with little or no disruption, as evidenced with the presence of fluid inclusions. An actively geysering body similar to modern Enceladus (Postberg et al., 2011) may be a reasonable analogue in this respect. Also, the originating body should have been within close proximity to the H chondrite parent in order to generate the number of halite grains seen in Monahans and Zag. Other candidates for Monahans/Zag halite parent bodie(s) may include a young Ceres with its possible liquid ocean, or Main Belt comets.

Zolensky, Michael E.↗