The SARS-CoV-2 virus causes COVID-19, and several of its gene products have been successfully targeted for antiviral drug development, including the 3C-like protease (3CLpro). The substrate-binding site of 3CLpro exhibits significant conformational plasticity. While available X-ray crystal structures reveal substantial loop variability and molecular dynamics simulations indicate that conformational heterogeneity persists in ligand-bound complexes, the slow-timescale thermodynamic and kinetic landscape of these complexes in solution remains incompletely defined. Using 19F NMR spectroscopy, we characterize a slow-exchange conformational equilibrium in both the covalent nirmatrelvir (NMV) and noncovalent ensitrelvir (ENS) complexes of 3CLpro. For the wild-type 3CLpro–NMV complex at 298 K, joint dual-field line shape and exchange spectroscopy analysis resolves a millisecond-timescale exchange between two distinct states (population ratio ∼ 65:35; kex ≈ 52 s−1; ΔG‡ ≈ 15 kcal mol−1). Co-existing bound states also persist in both the noncovalent NMV–[C145A] mutant (minor state population, pB ≈ 18%) and wild-type ENS (pB ≈ 38%) complexes, demonstrating that this slow-exchange heterogeneity is an intrinsic property of the ligated protease rather than a consequence of covalent attachment. These dynamics are not readily explained by available crystal structures or conventional microsecond molecular dynamics simulations, suggesting that in solution the inhibited enzyme samples alternative, energetically accessible conformations that are not fully represented in the crystal lattice. This study highlights the utility of solution-state 19F NMR for quantifying low-energy conformational states relevant to drug–target energetics and inhibitor design.
For the integration of two-dimensional materials in future devices, a fundamental understanding of their response to external stimuli is needed. Toward this goal, we have investigated the electron and spin dynamics in the metallic van der Waals material Fe3GeTe2 (FGT) in its paramagnetic state after ultrafast optical excitation. To this end, we have employed a zone plate streaking technique with probing energies in the extreme ultraviolet range, tuned to the Fe M2,3 and Te N4,5 absorption edges. This approach provides insights into energy-dependent charge dynamics with a sensitivity to transient absorption changes on the order of ∼ 10 - 4 . We find a slow carrier relaxation time at both elemental edges-up to ( 2.2 ± 0.6 ) ps in Te and exceeding several picoseconds in Fe-which is surprising for a metal. To elucidate the complex time-resolved response, we also employ static x-ray absorption spectroscopy at the corresponding elemental edges, in which we find a double feature at the Fe M2,3 edge. We attribute this to different Fe sites in the pristine material and an oxidized surface layer, and we propose that the time-resolved absorption dynamics show a mixture of signals stemming from the different species. Additionally, we conducted time-resolved x-ray magnetic circular dichroism measurements in FGT at room temperature. We do not find clear evidence of the previously observed light-induced ferromagnetic order above T C . Our study lays the groundwork for a deeper understanding of charge and spin dynamics in FGT after optical excitation as part of a roadmap for 2D spintronics.
Masatsune Kainosho (Kai) was invited to contribute to this volume, but shortly before his death on December 28, 2025, he sent an apologetic email stating that he was too ill to comply. This is the type of contribution Kai might have written had the fates of the subject and narrator been interchanged. Kai and I were mutual scientific admirers, collaborators, and great friends for 49 years. Our wives had common interests in weaving and other crafts. We enjoyed visiting one another, traveling together, staying in each other's homes, and getting to know family members. We shared interests in the good things in life as well as life's milestones of marriage, birth, and death.
Polysaccharide structural assignment via nuclear magnetic resonance (NMR) spectroscopy remains an analytical challenge due to spectral overlap because of limited chemical shift dispersion. This challenge is exacerbated by the wide-spread use of proton (1H) detection. Progress has also been hindered by the dispersed nature of carbohydrate databases and the restricted applicability of most prediction tools, which provide limited atom-specific residue discrimination in larger polymers. These limitations hamper the efficient characterization of glycosyltransferase (GT) activity, which depends on defining donor-acceptor substrate pairs and accurately identifying corresponding products. Here, we demonstrate the utility of high field 13C-detected NMR for the assignment of two homohexamers. Laminarihexaose and xylohexaose were analyzed using 2D heteronuclear correlation and correlation via long-range coupling (COLOC) experiments to evaluate whether complete residue level assignments could be achieved. The COLOC experiment revealed long range correlations that were not resolved using heteronuclear multiple bond correlation, and the 1D 13C spectra provided exceptional resolution, including distinct shoulders corresponding to residue specific chemical shift differences previously assumed to be indistinguishable. These findings suggest that high field 13C NMR can provide the nuanced atom level information required to train machine learning models for predicting chemical shift assignments of large, complex, and highly degenerate polysaccharides. Such models offer a promising framework for rapid structural identification of GT reaction products, enabling progress of high throughput characterization of plant derived polysaccharides central to renewable biomaterial development.
Strain engineering in epitaxial Bi(111) films on Si(111) enables temperature-tunable phonon frequencies through thermal expansion mismatch. Using in situ low-energy electron diffraction combined with broadband femtosecond transient reflectivity on the same samples, we directly correlate anisotropic lattice parameters with the redshift of the coherent A1g phonon mode. The in-plane lattice parameter remains locked to the Si substrate and is therefore nearly temperature-independent, imposing strong biaxial compressive strain, causing an additional expansion of the Bi lattice along the c-axis. By applying an extended phonon-shift model, we disentangle the contributions of intrinsic thermal expansion, thermal-mismatch strain, and anharmonicity to the phonon softening. The enhanced redshift observed in strained films cannot be accounted for by expansion or mismatch alone; instead, it arises from a strengthened anharmonic term that additionally scales with strain along the c-axis, being insensitive to the in-plane lattice parameter and thereby enabling targeted control of phonon frequencies.
This paper is a report of the High Data Rate Macromolecular Crystallography workshop held on 23 July 2025 as part of the 2025 meeting of the American Crystallographic Association in Lombard, IL, USA, 18-23 July 2025. This report summarizes the discussions, questions, action items, and recommendations that arose from the meeting and includes links to the presentations. The sessions were moderated by Aaron S. Brewster and Graeme Winter. There was particularly lively discussion about the possible need for lossy compression as data rates increase, as multimodal experiments become more popular and as research budgets are squeezed.
Bromodomains are conserved acetyl-lysine reader domains that play a central role in the assembly of transcriptional regulatory complexes. While generally presumed to function as monomers, bromodomain homo-dimers have been identified, and several bromodomain-containing proteins have been linked to biomolecular condensates, where locally elevated concentrations may promote dimerization. Here, we investigated bromodomain dimerization with an integrated approach that combines structural and biophysical measurements with AlphaFold-based predictions across the bromodomain family. Using the second bromodomain (BD2) of BRD4 as a model system, we characterized the thermodynamics and kinetics of its monomer-dimer equilibrium by two-dimensional nuclear magnetic resonance (NMR) lineshape analysis and CPMG relaxation dispersion. We found that the BRD4BD2 dimer forms transiently with a dissociation constant near 400 μM and a lifetime near 1 ms. Using our NMR-derived restraints, we performed data-driven docking to generate models of the BRD4BD2 dimer. To assess dimerization propensity across the wider bromodomain family, we leveraged AlphaFold-Multimer and AlphaFold3 to systematically predict homo-dimeric models for all human bromodomains. We identified several predicted dimer architectures, with 15 bromodomain dimers that have higher interface-confidence scores than BRD4BD2. Overall, our results suggest that weak and reversible dimerization may be more widespread among bromodomains, where it could contribute to function in dynamic transcriptional assemblies.
Combined histopathological and metabolomic analysis of the same tissue specimen can reduce biological variability compared to independent analyses of similar samples. However, conventional workflows are incompatible: tissue fixation leads to metabolite loss through dilution and chemical reactions with fixative, while metabolomic extraction typically destroys the specimen. We propose a method that reconciles these conflicting requirements by enabling metabolite recovery from fixative while preserving tissue for histopathology. In this approach, tissue is initially fixed in a small volume of fixative; an aliquot of the fixative is then collected for nuclear magnetic resonance-based metabolomic analysis before additional fixative is added for standard processing. As a proof-of-concept, mouse kidneys from the same subject were analyzed using two protocols: standard perchloric acid extraction (ensuring complete metabolite recovery but destroying tissue) and metabolite extraction from fixative. Several metabolites were fully recoverable from the fixative, others partially, while some were lost due to chemical reactions. Despite some limitations, this strategy may be useful for analyzing precious specimens, such as human biopsies, that must remain intact for further studies. Moreover, using the same specimen for dual analyses reduces the number of animals required and enhances statistical robustness.
A thermal neutron development beamline equipped with a Si monochromator has been commissioned at the HB-2D beam port of the High Flux Isotope Reactor at Oak Ridge National Laboratory. This instrument is dedicated to the development of neutron scattering methods and instrumentation, serving as a complement to the cold neutron development beamline CG-4B at the cold guide hall. Two incident wavelengths are available, 2.43 and 1.72 Å, with a flux of 1.75 and 1.28 ×105 n/(cm2 s), respectively. The instrument can be operated either in high-resolution or high-intensity mode through the horizontal bending of the monochromator. As the bending increases, the incident flux on the sample increases as a result of the additional lattice strain induced in the silicon wafers. With its mission centered on advancing neutron scattering instrumentation, the beamline has been named POPLAR, an acronym for polarized neutron development beamline for polarization analysis and Larmor labeling techniques.
The locus of enterocyte effacement-encoded regulator (Ler) is a master transcriptional activator essential for the virulence of enterohemorrhagic and enteropathogenic Escherichia coli. Although Ler shares homology with the global silencer H-NS, it functions uniquely as an anti-silencer, a role strictly dependent on its oligomerization state. However, the structural mechanism governing Ler assembly remains poorly understood. In this study, we have characterized the N-terminal oligomerization domain (Ler1-74) of Ler using solution NMR spectroscopy and biophysical assays, and found that Ler1-74 shows concentration-dependent oligomerization. We demonstrate that Ler oligomerization is driven by two distinct interfaces with contrasting dynamic properties. We determined the solution structure of the Ler18-74 dimer, revealing a stable, anti-parallel "tail-to-tail" interface (dimer Site-2, residues 35-66) stabilized by a hydrophobic core. In contrast, the N-terminal interface (dimer Site-1, residues 12-33) forms a highly dynamic "head-to-head" dimer, which undergoes significant conformational exchange and exhibits concentration- and temperature-dependent dimerization. Based on these findings, we propose a structural model wherein Ler forms supramolecular assemblies through the propagation of alternating stable (Site-2) and dynamic (Site-1) interactions. This architecture, while reminiscent of H-NS, displays distinct stability features that may underlie Ler's specific anti-silencing function in bacterial pathogenesis.
Bacterial conjugation facilitates horizontal gene transfer through the Type IV Secretion System (T4SS), a complex nanomachine central to antibiotic resistance dissemination. This study investigates the structure and dynamics of TraW, a key F-plasmid conjugative protein. TraW, in conjugation with the protein TrbC, is critical for F-pilus biogenesis and mating pair stabilization. Using biophysical, computational, and structural methods, including CD, NMR, SAXS, and native mass spectrometry, we characterize TraW as a modular protein with a stable C-terminal domain and a flexible N-terminal region. The full-length construct exhibits higher conformational adaptability and transient dimerization, whereas truncation enhances compactness and monomeric stability. AlphaFold modeling and SAXS analyses reveal that this flexibility, rather than intrinsic disorder, enables TraW to modulate inter-protein interactions essential for T4SS assembly and function. These findings establish TraW as a dynamic adaptor protein and highlight how flexibility fine-tunes structural plasticity in conjugative machinery.
Eldon Ulrich made fundamental contributions to the inception and development of the Biological Magnetic Resonance Bank (BMRB), the publicly accessible archive of biomolecualr data derived from the field of nuclear magnetic resonance (NMR) spectroscopy. He early on recognized the importance of properly referenced proton and carbon chemical shifts assigned to specific atoms in the chemical structure of a protein in delineating its three-dimensional structure. He pioneered the development of the data dictionary used by BMRB that captures precise information from the large array of different NMR experiments used in determining the structure, dynamics, and function of peptides, proteins, and nucleic acids. Eldon also developed a valuable branch of BMRB devoted to the NMR charicterization of metabolites, drugs, and other small molecules.
Understanding the structure and dynamics of hydrogen is critically important, yet direct experimental measurements are often challenging. Hydrogen interacts only weakly with common probing particles such as photons and electrons, and strong nuclear quantum effects can produce large nonthermal and anisotropic atomic displacements. Neutron scattering, however, provides a uniquely powerful approach due to the strong and distinct interactions of neutrons with atomic hydrogen, molecular hydrogen, and deuterium. Beyond neutron diffraction, which enables direct determination of hydrogen and deuterium positions, neutron vibrational spectroscopy—particularly when combined with computer simulations and modeling—offers unparalleled insights into hydrogen structure and dynamics that are inaccessible by other techniques. In this paper, after briefly summarizing the theoretical foundations, we review recent advances in applying neutron vibrational spectroscopy and computational methods to hydrogen-containing materials, ranging from molecular hydrogen adsorption to organic, inorganic, and hybrid compounds with diverse hydrogen local structure. Finally, we discuss opportunities offered by the recent progress in machine learning to further enhance the capabilities of this method.
Here, we investigate phonon mode- and electron band-selective electron-phonon couplings in centrosymmetric 1T'-MoTe2 using time- and angle-resolved photoemission spectroscopy combined with frequency-domain analysis. Femtosecond near-infrared pulses excite coherent A g -symmetric phonon modes at 2.34, 3.34, and 3.86 THz, which manifest as oscillatory modulations in photoemission intensity and binding energy across the valence bands. Pixel-wise Fourier analysis using recently developed methodologies reveals pronounced band selectivity with distinct coupling strengths for different electronic states and phonon modes, enabling the evaluation of band-renormalization amplitudes in the range of few meV. Ab initio calculations qualitatively reproduce the experimentally observed coupling patterns and relative trends, demonstrating the capability of combined experimental and theoretical approaches to resolve ultrafast electron-phonon interactions in quantum materials.
Ultrafast electron diffraction and phonon-diffuse scattering (UED(S)) experiments make use of photo-induced changes to electron scattering intensity across 2D detectors to report on a very wide range of dynamic structural phenomena in molecules and materials. Hybrid pixel counting detectors (HPCDs) are a promising technology for improved sensitivity and signal-to-noise in UED(S) experiments, as they offer near-zero readout noise and dark counts with the possibility of new acquisition modalities (e.g. shot-to-shot normalization) due to their high frame rates. However, it is well known that HPCDs suffer from count losses at high electron fluxes even in CW beam applications. How this translates to ultrashort electron pulse exposures has yet to be determined and is critical to understanding the application of this technology to ultrafast electron scattering experiments. Here we show that count losses are significantly exacerbated in ultrafast (pulsed) experiments, and that HPCDs require unconventional data handling and saturate above ≈2 electrons per pixel per pulse. This count-rate limitation presents a severe constraint on electron bunch charge when interrogating single crystal samples. Normalization strategies to optimize signal-to-noise in UED(S) and a complete model for measurement uncertainties using HPCDs are developed and tested using a large data set. Finally, we suggest ways HPCDs could be better adapted to ultrashort pulsed beam experiments.
Polyamines are polycations involved in both differentiation and proliferation of cells. Highly conserved polyamine biosynthetic enzymes are involved in the synthesis of polyamines. Spermidine synthase (SPDS) is an important enzyme in the polyamine biosynthetic pathway, and it is an aminopropyl-transferase that catalyzes the synthesis of the polyamine, spermidine, from putrescine and decarboxylated S-adenosine methionine. Spermidine has a variety of biological roles, including the formation of eIF5A, regulating autophagy, and stabilizing DNA and RNA. While there are numerous structures of human SPDS in complex with its substrates, products, or inhibitors, and numerous apo structures from various species, there is no structure of the apo form of human SPDS reported to date. In this study, the crystal structure of apo human SPDS was determined at 1.95 Å resolution. Comparison of the inherently flexible gatekeeping loop in the apo human structure with apo homologues revealed species-specific differences in loop conformation, indicating dynamics. Significant conformational change was observed in active site residues that are involved in catalysis when the apo human structure was compared to human ligand-bound complexes. These findings provide structural insights into the conformational dynamics and ligand-binding properties of spermidine synthase.
We present an investigation of one-photon valence-shell photoelectron spectroscopy and photoelectron circular dichroism (PECD) for the chiral molecule (1R,4R)-3-(heptafluorobutyryl)-(+)-camphor (HFC) and its europium complex Eu(III) tris[3-(heptafluorobutyryl)-(1R,4R)-camphorate] (Eu-HFC3), the latter of which constitutes the heaviest organometallic molecule for which PECD has yet been measured. We discuss the role of keto-enol tautomerism in HFC, both as a free molecule and complexed in Eu-HFC3. PECD is a uniquely sensitive probe of molecular chirality and structure such as absolute configuration, conformation, isomerization, and substitution, and is, in principle, well suited to unambiguously resolving tautomers; however, modeling remains challenging. For small organic molecules, theory is generally capable of accounting for experimentally measured PECD asymmetries, but significantly poorer agreement is typically achieved for the case of large open-shell systems. Here, we report PECD asymmetries, ranging up to ∼8% for HFC and ∼7% for Eu-HFC3, of similar magnitude to those reported previously for smaller isolated chiral molecules, indicating that PECD remains a practical experimental technique for the study of large, complicated chiral systems.
Protein structural information at the atomic resolution is indispensable in the search for lead compounds to develop therapeutic drugs for various diseases. To meet these demands, high-throughput analysis is of the utmost importance. X-ray crystallography, which can collect hundreds of datasets per day, is the best tool for rapidly obtaining protein structural information. To achieve rapid structure determination, we need to overcome two bottlenecks: crystallization and phasing. Here, we present our crystallization techniques and the MR-native SAD method using low-energy X-rays. Recently, even in the case of structural analysis of new proteins, it has become increasingly possible to determine the phases by the molecular replacement (MR) method using existing coordinates or structures obtained by AlphaFold2 as models. However, it is time-consuming to proceed with crystallographic refinement by gradually modifying the model and is not suitable for rapid structure determination. On the other hand, the MR-native SAD method, which combines the molecular replacement method and the native SAD method, can semi-automatically build a high-quality model from an electron density map calculated with the experimental SAD phases using the anomalous diffraction from sulfur. Furthermore, anomalous difference Fourier maps can be used to confirm the binding of compounds containing sulfur or phosphorus. In this presentation, we present our successful examples for the rapid determination of crystal structures [1-5].
