Time-resolved (TR) Raman spectroscopy is a unique tool for studying the dynamic properties of quantum matter and can become a key element of the multi-messenger research in the time domain. We present here the features and results of a novel setup for TR Raman, designed to expand the NFFA-SPRINT facility by integrating it with TR optical, transient grating and electron spectroscopy and spin polarization techniques. The TR Raman setup is characterized by a wide energy tunability of the pump and probe pulses, owing to the presence of a laser system providing ultrashort (50 fs to 2 ps) light pulses from the near ultraviolet to the infrared spectral regions. The ultra-high vacuum sample environment allows for the measurement of air-sensitive samples and ensures the full compatibility with photoelectron spectroscopies, as well as a wide sample temperature range. The functionalities of the setup and the multi-messenger research approach are here demonstrated by presenting studies of the relaxation dynamics in photoexcited semiconductor systems, namely, Si and MoS2. In addition, the pump-probe response of magnetite across the Verwey transition is presented, highlighting the capability of TR spontaneous Raman spectroscopy to be a valuable tool for probing photoinduced phase transitions in the time domain.

CRISPR-Cas [clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated] systems are adaptive immune systems present in bacteria and archaea to fight invading genetic elements. While proteins such as Cas9 and Cas12a cleave and inactivate the invader DNA, the mechanisms of which have been repurposed into efficient gene editing and gene therapy tools, Cas1 and Cas2 proteins are important in creating genetic memory of past invasions of foreign elements to create an active adaptive immune mechanism. Specifically, Cas1(4)-Cas2(2) complex (4:2 molar ratio of Cas1 and Cas2) integrates a piece of the invader DNA called the prespacer, sequence-specifically, into the CRISPR-Cas locus. The prespacer is transcribed to form CRISPR-RNA that guides Cas9/Cas12a, sequence-specifically, to the invader on secondary infection to inactivate the invader. While there are different types of CRISPR-Cas systems, Cas1-Cas2 proteins belonging to the type II-A CRISPR-Cas systems are unique since they can sequence-specifically integrate DNA into the CRISPR-Cas locus without additional protein factors, which is the case for other CRISPR types. This provides a unique opportunity to develop type II-A Cas1-Cas2 proteins as a site-specific integration tool for biotechnology and molecular biology applications. Previous studies from our lab have demonstrated that there are three different mechanisms within the type II-A systems for DNA integration that is based on protein-DNA sequence conservation and interactions. Specifically, a bioinformatics analysis of the “leader-repeat” junction of the CRISPR-Cas locus, a region where foreign DNA is integrated, showed three uniquely conserved junction sequences, named groups 1, 2, and 3 (G1, G2, G3). Cas1-Cas2 proteins also showed segregation mirroring that of the leader-repeat junction sequence, indicating coevolution of the DNA and protein elements. Bioinformatics results were further confirmed by in vitro integration assays that showed the tight dependence of the leader-repeat and protein elements of the cognate groups for successful integration. Of the different groups, G2 Cas1-Cas2 is very robust in integrating foreign DNA as an isolated system with the ability to insert diverse prespacer forms (e.g., overhang or splayed prespacer), compared to insertion of only very specific forms of the prespacer by the other two groups. This establishes G2 Cas1-Cas2 as a superior genome tool that has minimal requirements for sequence-specific DNA integration. To gain mechanistic insights on how G2 Cas1-Cas2 proteins accommodate different prespacers, we performed molecular dynamics simulations of G2 Cas1-Cas2 proteins bound to a prespacer and a target DNA holding the leader-repeat junction. We tested two different prespacers (overhang and splayed) with two different lengths of overhang/splaying (4 vs. 5). Our results show that, of the four Cas1 monomers, two have conformational changes in the binding site that is involved in catalyzing the prespacer integration. Specifically, two helices adopted a 3-10-helical conformation in the presence of the prespacers: one associated with the protein-DNA interaction at the leader junction and the other mediating the interactions with the divalent metal ion and the 3’-OH end of the prespacer in the active site . We propose that the presence of a 3-10-helix in key regions of active Cas1s may influence the stability of the prespacer-bound state. To understand the catalytic mechanism of DNA integration, we performed QM/MM simulations of Cas1- Cas2-prespacer-target DNA with divalent metal ions. Our results show that the number and arrangement of divalent metal ions at the active site significantly influence the structural configuration prior to catalysis. While previous research has shown a single divalent metal based catalysis, our results are hinting at the presence of a second potential Mg²+ binding site, suggesting a key role for metal coordination in substrate positioning, transition state stabilization, and overall enzymatic efficiency. To further dissect the reaction mechanism, we are currently investigating whether the DNA integration follows a stepwise or concerted pathway. Other ongoing studies include cryo electron microscopy-based structure determination and analysis of protein dynamics using hydrogen- deuterium exchange mass spectrometry (HDX-MS). The end goal is to integrate insights from these complementary approaches to assess the plasticity of the Cas1-Cas2 to accommodate different DNA substrates in their active sites. This information will enable fine-tuning of their use in biotechnological applications.

Since the Science nomination for “Breakthrough of the year 2018” [1], electron diffraction (3D ED, known also as micro-ED) for structural elucidation of organic compounds has been gaining a lot of momentum. 3D ED has been rapidly evolving as a complementary technique for SCXRD experiments [2]. Especially for those cases where single crystals of suitable size for XRD experiments are not obtained. Dedicated electron diffractometers [3] make the technique more accessible, resulting in many recent publications where these commercial electron diffractometers have been used [4].3D ED / Micro-ED’s ease of use and its potential for various applications is presented in multiple studies, especially in the pharmaceutical industry where it is having a high impact [5,6]. We would highlight some case studies where electron diffraction has helped the pharmaceutical industry obtain structural information not available before by other means [4b, 6]. Furthermore, we’ll delve and showcase other applications like: Characterization of meta-stable polymorphic forms, identifying crystallinity in amorphous solid dispersions, finding new polymorphic forms, identifying polymorphic forms for litigation purposes, impurities and phase identification using crystal mapping, and more.

Neutron diffraction provides a sophisticated, non-destructive means for the the atomic-resolution analysis of individual hydrogen atoms in enzymes. Here we describe how the precise location of protein and water hydrogen atoms using neutron diffraction provides a more complete description of the atomic and electronic structures of proteins, enabling key questions concerning enzyme reaction mechanisms, molecular recognition and binding and protein-water interactions to be addressed. We will briefly describe the capabilities that are available for neutron- based structural biology at ORNL, with special focus on the current upgrades and development of new instrumentation and software for neutron protein crystallographic analysis.

Pathogenic members of the genus Corynebacterium cause a wide range of serious infections in humans including diphtheria. Adhesion to host cells is a crucial step during infection. In Corynebacterium diphtheriae, adhesion is mediated by filamentous structures called pili or fimbriae that are covalently attached to the bacterial cell wall. Pilus assembly proceeds by transpeptidation reactions catalyzed by sortases, followed by covalent anchoring of the filament in the peptidoglycan layer.There are 6 sortases in C. diphtheriae, SrtA, SrtB, SrtC, SrtD, SrtE and SrtF. Five of these proteins are devoted to the assembly of three distinct types of pilus fibres: SrtA for the SpaA-type pilus, SrtB/SrtC for the SpaD-type pilus, and SrtD/SrtE for the SpaH-type pilus. SrtF, the so-called housekeeping sortase, catalyses the cell wall anchoring of pilin monomers as well as pili, but it does not polymerize pilins.We have previously determined structures of the SrtA and SrtF sortases and deposited to Protein Data Bank (PDB). There are no structures of remaining four C. diphtheriae sortases available. Also, biological data for these proteins are sparse. The presence of two sortase pairs involved in the SpaD-and SpaH-type pilus assembly is intriguing. The SrtB/SrtC sortases are able to polymerize SpaDF pili consisting of SpaD and SpaF proteins. However, only SrtB is capable of incorporation of SpaE accessory protein into the SpaD-type pili. There is even less data for SrtD/SrtE pair (40% amino acid identity between these proteins). It was shown that both proteins participate in formation of SpaHIG pili (SpaH-type pili including two accessory proteins SpaI and SpaG). Deletion of either one of sortases results in significantly slower polymerization of SpaH-type pili. No clear difference between SrtD and SrtE was reported.We crystallized and solved the high-resolution structures of four remaining C. diphtheriae sortases: SrtB, SrtC, SrtD and SrtE. The structures, with exception of SrtD, closely resemble Alphafold prediction models. We did not find significant differences between SrtB and SrtC sortase main bodies and active centers. The major difference between SrtB and SrtC is the presence SrtB extended proline-rich 18-residue loop replacing short 3-residue fragment in the SrtC. We think this loop is indirectly involved in the SrtB activity. Also, for SrtD and SrtE sortase pair, we did not find significant differences in core structures and active centers. Nevertheless, we found a unique conformation of the N-terminal helix not predicted by Alphafold models. The helix of the experimental structure is rotated by 180 degrees relatively to the protein main body. We do not exclude possibility that it is a result of crystal packing. Nevertheless, it shows mobility /flexibility of this SrtD region. We think that our structural data and mutagenesis of sortases will help to elucidate differences in sortase pairs and will help understand their biological purpose.

G protein-coupled receptors (GPCRs) are essential transmembrane proteins playing key roles in human health and disease. Understanding their atomic-level molecular structure and conformational states is imperative for advancing drug development. Recent breakthroughs in single-particle cryo-EM have propelled the structural biology of GPCRs into a new era. Nevertheless, the preparation of suitable GPCR samples and their complexes for cryo-EM analysis remains challenging due to their poor stability and highly dynamic nature. Here, we present our online buffer exchange-native MS method combined with Direct Mass Technology (OBE-nMS+DMT) which facilitates high-throughput analysis and guides sample preparation. We applied this method to optimize the GPR119-Gs complex sample prior to cryo-EM analysis, leading to a 3.51 Å resolution structure from only 396 movies collected on a 200 kV Glacios. This study suggests that the OBE-nMS+DMT method emerges as a powerful tool for prescreening sample conditions in cryo-EM studies of GPCRs and other membrane protein complexes.

High-entropy alloys (HEAs) are a new class of alloys with great potential for harsh environment applications. A precipitation-hardened HEA, (Fe0.3Ni0.3Mn0.3Cr0.1)88Ti4Al8, was developed and produced via conventional manufacturing with high strength but low ductility, due to an extensive network of brittle intermetallics that formed after prolonged artificial aging. Additive manufacturing (AM) can often drastically change the microstructure and properties of alloys, so this HEA was also printed using laser powder bed fusion. To induce precipitation and study its kinetics, the printed alloy was subjected to artificial aging at three different temperatures. The aging process was observed in-situ under synchrotron XRD and characterized after aging by SEM, EBSD, and TEM. Results indicated that the as-printed alloy exhibited excellent tensile properties due to its fine grain size and precipitation during printing. Aging induced additional precipitation, especially of the ordered L12 phase. This additional precipitation occurred more quickly than aging precipitation in the conventional manufactured sample, and some precipitates showed a preference for grain boundary nucleation.

Extracellular ATP serves as a crucial signaling molecule, present in varying concentrations across diverse cellular environments. The P2X receptor (P2XR) family, which recognizes extracellular ATP, consists of seven subtypes (P2X1R - P2X7R) that form functional homo- and hetero- trimeric ion channels. These receptors are activated by distinct concentrations of extracellular ATP, ranging from low nanomolar to high micromolar levels, and are expressed in numerous cell types. They play key roles in a variety of pathophysiological conditions affecting the central nervous, immune, and cardiovascular systems. Beyond differences in ATP sensitivity, the kinetics of ion channel gating in response to agonists vary significantly among P2XR subtypes. For example, P2X1Rs and P2X3Rs exhibit rapid desensitization (milliseconds), while P2X2Rs and P2X4Rs undergo slower desensitization (seconds), and P2X7Rs show little to no desensitization. Although membrane-proximal regions within the cytoplasmic termini have long been known to influence P2XR desensitization, the lack of structural data for these domains in any P2XR subtype hindered the development of a detailed molecular explanation for the diverse desensitization profiles. Furthermore, the lack of desensitization observed in P2X7R remained particularly puzzling. In 2016, the first crystal structures of P2X3R in three functional conformational states, including a desensitized state, revealed a novel desensitization mechanism, termed the “helical recoil” model. Later, in 2019, Cryo-EM structures of full-length P2X7R not only confirmed this model but also provided additional insight into the mechanisms that prevent desensitization in P2X7Rs. Recent Cryo-EM structures of the human P2X4R not only refine the helical recoil model but also identify a putative lipid-binding site in the cytoplasmic domain that further modulates desensitization kinetics. Over the past decade, structural biology has significantly advanced our understanding of P2XR structure and function, particularly in relation to the molecular mechanisms of receptor gating.

Electrochemical energy storage is an enabling technology for personal and industrial electronics, adoption of intermittent renewable energy, and the electrification of transportation. From a fundamental solid-state chemistry perspective, and in the context of batteries, it is interesting to explore new mixed ionic–electronic conductors that can withstand large changes in composition and electronic configuration over ∼1000 charge– discharge cycles to function as electrode materials and to explore new pure ion conductors with extremely low electronic conductivities that could function as solid electrolytes or interfacial coatings. Understanding the mechanisms that facilitate ion and/or electron transport or induce material degradation are the keys to discovering and engineering the next generation of battery materials.Relatively few unique crystal structures underpin most battery materials. We are particularly interested in novel complex oxides that might offer new insights into structure–property relationships or even new performance characteristics. This talk will focus on recent examples from our lab: (i) defects, electrochemistry, and metal–metal bonding in NaNb7O18 and NaNb13O33 framework structures (Wadsley–Roth derivatives) with tunnel-blocking defects, and (ii) new lithium-rich “layered” structures, Li3MO4 (M = Nb, Ta) synthesized via instantaneous ion exchange in a molten salt flux. Both families of materials are characterized with an ‘NMR crystallography’ approach that combines X-ray and neutron diffraction with DFT-supported solid-state NMR spectroscopy.

Crystallography has a wide range of applications in research, but this field is rarely discussed in primary and secondary school classrooms. While it is difficult to do cutting-edge, cancer-curing research in a high school laboratory, the intricacies of crystallography can still be introduced to students of any age. Through the Structural Nucleic Acid Anticancer Research Society (STARS), we have been able to help provide educational experiences and opportunities with crystal-growing and crystallography for students grades K-12th.At the high school level, STARS has organized weekly club meetings to work with students in growing and experimenting with inorganic crystals. Students choose the best, most unique crystals of the ones they have grown to submit to the U.S. Crystal Growing Competition. Having this opportunity provides students with foundational knowledge of crystallography and can excite students about science, thus inspiring a new generation of scientists.Beyond weekly meetings, high school STARS has also worked in conjunction with the collegiate STARS branch to offer crystallography workshops and camps. Through the workshops we provide students with the opportunity to experience wet lab research with lysozyme and gain a greater understanding of crystallography and its applications. Through the summer camp, which we hosted July 1-4, 2024, we allowed younger students to grow their own inorganic crystals and learn more about the science behind crystallography and its applications. Not only was it an educational experience for the students attending the camp, but it also a valuable opportunity for members of the high school STARS group to practice public speaking and science communication, both of which are valuable skills in a variety of professions.STARS will continue to provide valuable lab experience through club meetings, workshops, camps, and more. We hope to continue to provide K-12th students with educational experiences that will help inspire the young scientists that are the future of this field of study.