BioSciences

The structural biology method of X-ray footprinting mass spectrometry (XFMS) is available at two national synchrotron beamlines in the USA: one at the Advanced Light Source (ALS) on the West Coast and the other at the National Synchrotron Light Source II on the East Coast. XFMS is a solution-state technique that utilizes oxidative modifications of proteins at micromolar concentrations in aqueous buffer to extract structural information. X-rays are employed to generate hydroxyl radicals in situ, which covalently modify specific protein side chains. These modifications are subsequently quantified using liquid chromatography and mass spectrometry. Ratiometric changes in modification levels between two protein states (e.g. with and without ligand) generate a relative solvent accessibility map of the protein pairs, which serves to reveal structural features. Up until recently, the XFMS capability was available as part of a shared program at the ALS without a dedicated beamline. In this article, we describe the commissioning of ALS beamline 3.3.1, dedicated to XFMS, including the installation of a new focusing mirror, the design and construction of a new endstation with automated sample handling and exposure capabilities, and the use of accurate empirical dose calculations using Gafchromic film. Finally, we showcase the new beamline capabilities using two protein systems.

Serial femtosecond crystallography (SFX) and continuous serial electron diffraction (c-SerialED) both enable high-resolution structure determination from protein microcrystals with minimal radiation damage, making it ideal for studying redox-active metalloenzymes. Here, c-SerialED and SFX were used to solve structures of the class Ia ribonucleotide reductase R2 subunit in oxidized (Fe^III-Fe^III), reduced (Fe^II-Fe^II), and re-oxidized states at ∼1.8 Å resolution, capturing three points in a redox reaction. These results demonstrate that c-SerialED can track reversible changes at the redox-site, enabling future time-resolved studies. Comparison between c-SerialED structures and SFX diffraction and emission data confirmed minimal radiation damage. Furthermore, previously reported structures use mercury in the crystallization condition and show mercury-induced conformational changes. Here, we use mercury-free crystallization conditions and reveal a water molecule in the redox center of the reduced state, absent in the previous structures, making these structures more representative of the physiological state.

Lipid nanoparticles (LNPs) are versatile carriers for nucleic acid (NA) therapeutics, including ASOs, siRNA, mRNA, and poly-IC. While lipid composition is known to influence LNP properties, the impact of NA length on morphology and internal structure is less understood, particularly during the stages of carrier-cargo assembly. Here, we examine NA length and lipid composition immediately after mixing using high-throughput SAXS, dynamic light scattering, and cryogenic electron microscopy. All LNPs form ordered NA/lipid compartments, with longer NAs promoting inverse hexagonal (H_II) phases and larger intercompartment distances. In contrast, short NAs, especially in formulations with SM102 ionizable lipid, favor lamellar phases. SAXS peak deconvolution quantifies ordered versus disordered phases via a Robustness of Ordered Phase factor, which correlates with particle size and encapsulation efficiency. Formulations with MC3 ionizable and DOPE helper lipids exhibit the most stable H_II-phase packing, highlighting the role of helper-lipid curvature in compartment stabilization. Variations in NA compartmentalization indicate differences in payload capacity, offering a framework for rational LNP design across diverse nucleic acid cargos.

Pancreatic beta cells contain insulin secretory granules (ISGs), organelles where proinsulin is converted into insulin. As ISGs mature, they undergo extensive biophysical remodeling, producing a spectrum of subpopulations with heterogeneous molecular and spatial characteristics. However, systematic methods to define ISG subpopulations remain underdeveloped. To address this gap in knowledge, we employed soft X-ray tomography (SXT), which can quantitatively measure the biochemical density of ISGs within whole beta cells. Using unsupervised clustering, we classified subpopulations based on molecular density, size, and spatial positioning. Across different insulin secretory stimuli, we observed shifts toward mature and releasable subtypes, demonstrating that exogenous signals can dynamically remodel ISG subpopulation distributions. We extended this methodology to primary beta cells characterized using volume electron microscopy (vEM). Integrating subpopulations from SXT and vEM uncovered insights inaccessible by a single method in isolation. This strategy establishes a framework for defining therapeutic approaches aimed at enriching physiologically beneficial ISG subpopulations.

Protein oxidative footprinting, using hydroxyl radical labeling detected by bottom-up proteomics, has progressed from an emerging method to a widely used approach in structural biology. Hydroxyl radicals generated from hydrogen peroxide (via photolysis, Fenton chemistry or electrochemistry) or directly from water (via X-rays, plasma or gamma rays) irreversibly encode structural information within protein side chains, which is read out using standard liquid chromatography–mass spectrometry workflows. Quantitative changes in labeling report on solvent accessibility and reveal effects of protein–protein interactions, ligand binding, protein folding, conformational changes or applied stress. Comparing labeling patterns between states provides detailed maps of structural changes and interaction sites. Over the past decade, oxidative footprinting has proven valuable as a solution-phase and in-cell method for protein structure analysis. This Perspective summarizes best practices for experimental design, sample processing, data analysis, interpretation and integration with orthogonal data, offering a consensus framework to guide application of oxidative footprinting in academic and biopharmaceutical research.

We report the successful resolution of the multiplet structure of Kα1 x-ray emission in manganese (Mn) complexes through seeded stimulated x-ray emission spectroscopy (seeded S-XES). Using a femtosecond pump pulse above the Mn K edge to generate simultaneous 1s core holes, and a second-color tunable seed pulse to initiate the stimulated emission process, we were able to enhance individual lines within the Kα1 emission. This approach allows to resolve the fine multiplet features that are obscured by lifetime broadening in conventional (spontaneous) Mn Kα XES. The work builds on our previous observation that S-XES from Mn(II) and Mn(VII) complexes pumped at high intensities can exhibit stimulated emission without sacrificing the chemical sensitivity to oxidation states. This technique opens the door to controlled high-resolution electronic structure spectroscopy in transition-metal complexes beyond the core-hole lifetime, with potential applications in catalysis, inorganic chemistry, and materials science.

Triticum turgidum cv Kronos is a tetraploid wheat cultivar that underpins one of the most widely used community platforms for functional genomics. Over the past decade, researchers have generated c. 3000 exome-capture (EC) and promoter-capture (PC) datasets linked to mutagenized seed stocks, along with extensive transcriptomic and phenotypic resources. However, the absence of a reference genome has constrained their full utility. We assembled a chromosome-scale reference genome for Kronos, with high-confidence annotations, including manual curation of over 1000 disease resistance (nucleotide-binding leucine-rich repeat (NLR)) genes and genome-wide identification of microRNAs and phasiRNAs. We additionally reanalyzed EC and PC data to capture mutational landscapes across ethyl methane sulphonate-mutagenized Kronos populations. We revealed previously hidden NLR diversity and resolved their genomic organization at chromosomal ends. Re-analysis of capture datasets enabled high-resolution mutation discovery in genes and regulatory regions, providing a more comprehensive view of the variations detectable in the Kronos mutant populations. Collectively, these resources provide a reference-quality genomic framework for Kronos and position it as a versatile platform for functional and translational wheat research.

The biological small-angle X-ray scattering (bioSAXS) beamline BM29 at the ESRF, operated by the ESRF-EMBL Joint Structural Biology and bioImaging Group (JSBIG), resumed user operation in September 2020 following the ESRF extremely brilliant source (EBS) upgrade. To exploit the high quality of X-ray beam delivered by this new fourth-generation synchrotron source, BM29 underwent significant refurbishment, including source optimization, instrument upgrades, and a complete redesign of the whole experimental control system. Here, we introduce the BioSAXS Customized Beamline Environment (BSxCuBE-Web), a new open-source, web-based platform designed to streamline, automate and enhance bioSAXS data collection. BSxCuBE-Web offers an intuitive and user-friendly interface for expert and non-expert users alike, to easily define and run bioSAXS experiments, as well as to monitor both raw and processed data. Its adaptable architecture facilitates its deployment on beamlines beyond the ESRF. In 2025 alone, BSxCuBE-Web supported more than 97 experiments involving over 256 users at BM29. The interface has been successfully validated using a wide range of biological samples and continues to be actively developed in response to bioSAXS user feedback and evolving experimental requirements. User experience reports have been overwhelmingly positive, highlighting significant improvements in efficiency, accessibility, and reliability during data collection.

Time-resolved step-scan Fourier transform infrared (FTIR) difference spectroscopy was used to obtain (A₁ ^- - A₁) FTIR difference spectra from photosystem I (PSI) samples isolated from eight phylogenetically diverse cyanobacterial strains and one green alga, totaling 13 PSI preparations. These included samples from cells grown under far-red light and PSI in monomeric, dimeric, trimeric, and tetrameric states. Spectral profiles were shown to be independent of oligomeric state. Remarkably, all (A₁ ^- - A₁) FTIR difference spectra exhibited high similarity, underscoring the robustness of the technique and indicating minimal experimental variability. This congruence reveals a highly conserved environment for the phylloquinone cofactor at the A₁ binding site across diverse taxa. Conserved bands associated with the A₀ pigment further suggest structural continuity from A₀ to A₁. To leverage this consistency, we constructed a composite (A₁ ^- - A₁) FTIR difference spectrum by averaging all 13 spectra. This composite spectrum provides enhanced resolution, enabling unambiguous identification of previously unresolved bands. The fact that a highly resolved composite spectrum can be obtained by averaging demonstrates the similarity in the spectra from the different types of samples. Band assignments were refined using prior studies, yielding an improved spectral framework for future investigations of PSI electron transfer cofactors.