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SEL Inc., Juniper Networks and Dragos Networks, in association with our industry partners Bonneville Power Administration (BPA) and New York Power Authority (NYPA) (collectively: the Parties), have completed contractual agreement DOE-0000900 with U.S. Dept. of Energy developing next generation automation, analytics and security solution architectures that leveraged.
Cybersecurity companies such as CrowdStrike, Dragos, Microsoft and Unit 42 categorize Advanced Persistent Threats (APTs) using their own naming schemes. As a result, these APTs are mapped to different malware sources and campaigns, all from differing sources, leading to inconsistent mapping. Inconsistent mapping causes confusion and adds further obscurity around these groups, making it difficult to track and mitigate APT cyberattacks. The Evidence-based Graph Adversary Mapping (EGRAM) tool remediates the mapping challenge by collecting, updating and converting adversary data and their sources into a valid, codified STIX v2.1 bundle which is then stored in a Neo4j graph database. It utilizes graph traversal methods and centrality analysis to generate actionable information as a Structured Threat Intelligence Graph (STIG), based on user queries. EGRAM exists as Python code and a Jupyter Notebook that acts as a searchable, evidence-based, source of intelligence for APT groups’ artifacts and cyber campaigns.
Overhauser effect (OE) dynamic nuclear polarization (DNP) has drawn attention owing to its enhanced performance at ultrahigh magnetic fields. The lack of design principles has, nevertheless, limited the development of OE polarizing agents when compared to those used for cross-effect DNP. Here, we measured the 19 F OE DNP performance of a series of CF 3 -functionalized Blatter-type radicals. Using density functional theory calculations, we accurately predict the methyl-driven OE performance, paving the way for computer-aided design of optimized OE DNP polarizing agents.
Abstract Biologically active vitamin B 6 -derivative pyridoxal 5′-phosphate (PLP) is an essential cofactor in amino acid metabolic pathways. PLP-dependent enzymes catalyze a multitude of chemical reactions but, how reaction diversity of PLP-dependent enzymes is achieved is still not well understood. Such comprehension requires atomic-level structural studies of PLP-dependent enzymes. Neutron diffraction affords the ability to directly observe hydrogen positions and therefore assign protonation states to the PLP cofactor and key active site residues. The low fluxes of neutron beamlines require large crystals (≥0.5 mm 3 ). Tryptophan synthase (TS), a Fold Type II PLP-dependent enzyme, crystallizes in unit gravity with inclusions and high mosaicity, resulting in poor diffraction. Microgravity offers the opportunity to grow large, well-ordered crystals by reducing gravity-driven convection currents that impede crystal growth. We developed the Toledo Crystallization Box (TCB), a membrane-barrier capillary-dialysis device, to grow neutron diffraction-quality crystals of perdeuterated TS in microgravity. Here, we present the design of the TCB and its implementation on Center for Advancement of Science in Space (CASIS) supported International Space Station (ISS) Missions Protein Crystal Growth (PCG)-8 and PCG-15. The TCB demonstrated the ability to improve X-ray diffraction and mosaicity on PCG-8. In comparison to ground control crystals of the same size, microgravity-grown crystals from PCG-15 produced higher quality neutron diffraction data. Neutron diffraction data to a resolution of 2.1 Å has been collected using microgravity-grown perdeuterated TS crystals from PCG-15.
The global effects of climate change will increase the frequency and intensity of extreme events such as heatwaves and power outages, which have consequences for buildings and their cooling systems. Buildings and their cooling systems should be designed and operated to be resilient under such events to protect occupants from potentially dangerous indoor thermal conditions. This study performed a critical review on the state-of-the-art of cooling strategies, with special attention to their performance under heatwaves and power outages. We proposed a definition of resilient cooling and described four criteria for resilience—absorptive capacity, adaptive capacity, restorative capacity, and recovery speed —and used them to qualitatively evaluate the resilience of each strategy. The literature review and qualitative analyses show that to attain resilient cooling, the four resilience criteria should be considered in the design phase of a building or during the planning of retrofits. The building and relevant cooling system characteristics should be considered simultaneously to withstand extreme events. A combination of strategies with different resilience capacities, such as a passive envelope strategy coupled with a low-energy space-cooling solution, may be needed to obtain resilient cooling. Finally, a further direction for a quantitative assessment approach has been pointed out.
The assembly of two monomeric constructs spanning segments 1-199 (MPro 1-199 ) and 10-306 (MPro 10-306 ) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ~7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro 1-199 and MPro 10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201 to 214 onto a second helical domain of MPro 10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro 1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro 10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ~6 μM MPro 1-199 . Catalytic activity arises primarily from MPro 1-199 and is dependent on the interface interactions involving the N-finger residues 1 to 9 of MPro 1-199 and E290 of MPro 10-306 . Importantly, our results confirm that a single N-finger region with its associated intersubunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.
Pyridoxal 5′-phosphate (PLP), the biologically active form of vitamin B6, is an essential cofactor in many biosynthetic pathways. The emergence of PLP-dependent enzymes as drug targets and biocatalysts, such as tryptophan synthase (TS), has underlined the demand to understand PLP-dependent catalysis and reaction specificity. The ability of neutron diffraction to resolve the positions of hydrogen atoms makes it an ideal technique to understand how the electrostatic environment and selective protonation of PLP regulates PLP-dependent activities. Facilitated by microgravity crystallization of TS with the Toledo Crystallization Box, we report the 2.1 Å joint X-ray/neutron (XN) structure of TS with PLP in the internal aldimine form. Positions of hydrogens were directly determined in both the α- and β-active sites, including PLP cofactor. The joint XN structure thus provides insight into the selective protonation of the internal aldimine and the electrostatic environment of TS necessary to understand the overall catalytic mechanism.
SARS-CoV-2 propagation under nirmatrelvir and ensitrelvir pressure selects for main protease (MPro) drug-resistant mutations E166V (DRM2), L50F/E166V (DRM3), E166A/L167F (DRM4), and L50F/E166A/L167F (DRM5). DRM2-DRM5 undergoes N-terminal autoprocessing to produce mature MPro with dimer dissociation constants (K dimer ) 2–3 times larger than that of the wildtype. Co-selection of L50F restores catalytic activity of DRM2 and DRM4 from ~10 to 30%, relative to that of the wild-type enzyme, without altering K dimer . Binding affinities and thermodynamic profiles that parallel the drug selection pressure, exhibiting significant decreases in affinity through entropy/enthalpy compensation, were compared with GC373. Reorganization of the active sites due to mutations observed in the inhibitor-free DRM3 and DRM4 structures as compared to MPro WT may account for the reduced binding affinities, although DRM2 and DRM3 complexes with ensitrelvir are almost identical to MPro WT -ensitrelvir. In conclusion, chemical reactivity changes of the mutant active sites due to differences in electrostatic and protein dynamics effects likely contribute to losses in binding affinities.
The Overhauser effect is unique among DNP mechanisms in that it requires the modulation of the electron-nuclear hyperfine interactions. While it dominates DNP in liquids and metals, where unpaired electrons are highly mobile, Overhauser DNP is possible in insulating solids if rapid structural modulations are linked to a modulation in hyperfine coupling. In this study, we report that Overhauser DNP can be triggered by the strategic addition of a methyl group, demonstrated here in a Blatter’s radical. The rotation of the methyl group leads to a modulation of the hyperfine coupling to its protons, which in turn facilitates electron-nuclear cross-relaxation. Removal of the methyl protons, through deuteration, quenches the process, as does the reduction of the hyperfine coupling strength. This result suggests the possibility for the design of tailor-made Overhauser DNP polarizing agents for high-field MAS-DNP.
Recently discovered 2D van der Waals magnetic materials, and specifically iron–germanium–telluride (Fe5GeTe2), have attracted significant attention both from a fundamental perspective and for potential applications. Key open questions concern their domain structure and magnetic phase transition temperature as a function of sample thickness and external field, as well as implications for integration into devices such as magnetic memories and logic. Here we address key questions using a nitrogen-vacancy center based quantum magnetic microscope, enabling direct imaging of the magnetization of Fe5GeTe2 at submicrometer spatial resolution as a function of temperature, magnetic field, and thickness. This quantum imaging technique provides noninvasive, high-sensitivity measurements with high spatial resolution under ambient conditions, making it particularly well suited for probing 2D magnets. We employ spatially resolved measures, including magnetization variance and cross-correlation, and find a significant spread in transition temperature yet with no clear dependence on thickness down to 15 nm. We also identify previously unknown stripe features in the optical as well as magnetic images, which we attribute to modulations of the constituting elements during crystal synthesis and subsequent oxidation. Our results suggest that the magnetic anisotropy in this material does not play a crucial role in their magnetic properties, leading to a magnetic phase transition of Fe5GeTe2 which is largely thickness-independent down to 15 nm. Our findings could be significant in designing future spintronic devices, magnetic memories, and logic with 2D van der Waals magnetic materials.
In exsolution, nanoparticles form by emerging from oxide hosts by application of redox driving forces, leading to transformative advances in stability, activity, and efficiency over deposition techniques, and resulting in a wide range of new opportunities for catalytic, energy and net-zero-related technologies. However, the mechanism of exsolved nanoparticle nucleation and perovskite structural evolution, has, to date, remained unclear. Herein, we shed light on this elusive process by following in real time Ir nanoparticle emergence from a SrTiO 3 host oxide lattice, using in situ high-resolution electron microscopy in combination with computational simulations and machine learning analytics. We show that nucleation occurs via atom clustering, in tandem with host evolution, revealing the participation of surface defects and host lattice restructuring in trapping Ir atoms to initiate nanoparticle formation and growth. These insights provide a theoretical platform and practical recommendations to further the development of highly functional and broadly applicable exsolvable materials.
Nutrient foraging by fungi weathers rocks by mechanical and biochemical processes. Distinguishing fungal-driven transformation from abiotic mechanisms in soil remains a challenge due to complexities within natural field environments. We examined the role of fungal hyphae in the incipient weathering of granulated basalt from a three-year field experiment in a mixed hardwood-pine forest (S. Carolina) to identify alteration at the nanometer to micron scales based on microscopy-tomography analyses. Investigations of fungal-grain contacts revealed (i) a hypha-biofilm-basaltic glass interface coinciding with titanomagnetite inclusions exposed on the grain surface and embedded in the glass matrix and (ii) native dendritic and subhedral titanomagnetite inclusions in the upper 1–2 µm of the grain surface that spanned the length of the fungal-grain interface. We provide evidence of submicron basaltic glass dissolution occurring at a fungal-grain contact in a soil field setting. An example of how fungal-mediated weathering can be distinguished from abiotic mechanisms in the field was demonstrated by observing hyphal selective occupation and hydrolysis of glass-titanomagnetite surfaces. We hypothesize that the fungi were drawn to basaltic glass-titanomagnetite boundaries given that titanomagnetite exposed on or very near grain surfaces represents a source of iron to microbes. Furthermore, glass is energetically favorable to weathering in the presence of titanomagnetite. Our observations demonstrate that fungi interact with and transform basaltic substrates over a three-year time scale in field environments, which is central to understanding the rates and pathways of biogeochemical reactions related to nuclear waste disposal, geologic carbon storage, nutrient cycling, cultural artifact preservation, and soil-formation processes.
Pyridoxal 5’-phosphate (PLP)-dependent enzymes utilize a vitamin B 6 -derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of small-molecule antimicrobials and anticancer therapeutics, while others have been used to synthesize pharmaceutical building blocks. Understanding PLP-dependent catalysis and the reaction specificity is crucial to advance structure-assisted drug design and enzyme engineering. Here we report the direct determination of the protonation states in the active site of Thermus thermophilus SHMT (TthSHMT) in the internal aldimine state using room-temperature joint X-ray/neutron crystallography. Conserved active site architecture of the model enzyme TthSHMT and of human mitochondrial SHMT (hSHMT2) were compared by obtaining a room-temperature X-ray structure of hSHMT2, suggesting identical protonation states in the human enzyme. The amino acid substrate serine pathway through the TthSHMT active site cavity was tracked, revealing the peripheral and cationic binding sites that correspond to the pre-Michaelis and pseudo-Michaelis complexes, respectively. At the peripheral binding site, the substrate is bound in the zwitterionic form. By analyzing the observed protonation states, Glu53, but not His residues, is proposed as the general base catalyst, orchestrating the retro-aldol transformation of L-serine into glycine.
Pyridoxal 5'-phosphate (PLP)-dependent enzymes have been extensively studied for their ability to fine-tune PLP cofactor electronics to promote a wide array of chemistries. Neutron crystallography offers a straightforward approach to studying the electronic states of PLP and the electrostatics of enzyme active sites, responsible for the reaction specificities, by enabling direct visualization of hydrogen atom positions. Here we report a room-temperature joint X-ray/neutron structure of aspartate aminotransferase (AAT) with pyridoxamine 5'-phosphate (PMP), the cofactor product of the first half reaction catalyzed by the enzyme. Between PMP N SB and catalytic Lys258 Nζ amino groups an equally shared deuterium is observed in an apparent low-barrier hydrogen bond (LBHB). Density functional theory calculations were performed to provide further evidence of this LBHB interaction. The structural arrangement and the juxtaposition of PMP and Lys258, facilitated by the LBHB, suggests active site preorganization for the incoming ketoacid substrate that initiates the second half-reaction.
Neutron crystallography revealed protonation states in Tth SHMT-FA complex. Glu53 is protonated but other residues maintain protonation states upon FA binding. Structural analyses support key roles of Glu53 and gating loop dynamics in SHMT function.
Serine hydroxymethyltransferase (SHMT) is a pyridoxal-5′-phosphate (PLP) dependent enzyme that catalyzes a chemical transformation essential for the one-carbon (1C) metabolism. SHMT reversibly converts L-Ser into Gly and transfers a 1C unit to tetrahydrofolate (THF) to give 5,10-methylene-THF (5,10-MTHF). 5,10-MTHF, a 1C-unit donor, plays a crucial role in the downstream biomolecular syntheses required for the cell homeostasis and proliferation. SHMT is a prominent target for the drug discovery to battle bacterial and parasitic infections, and to treat various types of cancer. SHMT-catalyzed chemistry is governed by the general acid-base catalysis. Knowledge of the catalytic mechanism can aid drug design but can only be achieved when the atomic details of each reaction step are mapped, including accurate determination of hydrogen atom positions. Here we utilized the inactive E53Q mutant of Thermus thermophilus ( Tth ) SHMT to directly determine protonation states with room-temperature neutron crystallography and to capture a reactive intermediate containing the PLP-L-Ser external aldimine and THF in the enzyme active site. We observed protonation of the Schiff base nitrogen (N SB ) in the PLP internal aldimine but no change in the protonation states of other ionizable PLP groups and active site residues compared to wild-type Tth SHMT. X-ray structural analysis of the ternary intermediate complex E53Q-Ser-THF that eluded previous structural characterization shows the strategic positioning of the E53Q side chain in close proximity to the external aldimine and THF and reinforces the proposed role for E53 as the driver of proton transfer events along the reaction pathway.
The Overhauser effect in the dynamic nuclear polarization (DNP) of non-conducting solids has drawn much attention due to the potential for efficient high-field DNP as well as a general interest in the underlying principles that enable the Overhauser effect in small molecules. We recently reported the observation of 1 H and 2 H Overhauser effects in H 3 C- or D 3 C-functionalized Blatter radical analogs, which we presumed to be caused by methyl rotation. In this work, we look at the mechanism for methyl-driven Overhauser DNP in greater detail, considering methyl librations and tunneling in addition to classical rotation. Here, we predict the temperature dependence of these mechanisms using density functional theory and spin dynamics simulations. Comparisons with results from ultralow-temperature magic angle spinning-DNP experiments revealed that cross-relaxation at temperatures above 60 K originates from both libration and rotation, while librations dominate at lower temperatures. Due to the zero-point vibrational nature of these motions, they are not quenched by very low temperatures, and methyl-driven Overhauser DNP is expected to increase in efficiency down to 0 K, predominantly due to increases in nuclear relaxation times.
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.