I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[38313]
Open Access
Abstract: Radical enzymes, including glycyl radical enzymes (GREs) and B12-dependent enzymes, catalyze a wide range of biochemical transformations through radical-based mechanisms. An unusual property—conditional resistance to chymotrypsin digestion—has previously been reported for two GREs. However, whether this feature is broadly conserved among related radical enzymes and what factors trigger it has remained unclear. In this study, we investigated five radical enzymes: four GREs and one B12-dependent diol dehydratase. Proteolytic assays demonstrated that substrate binding significantly enhances resistance to chymotrypsin degradation, suggesting a conserved conformational shift from an open, protease-sensitive state to a closed, protease-resistant form. X-ray crystallographic analysis of a GRE-type 1,2-propanediol dehydratase from Raoultella planticola confirmed that active site occupancy correlates with increased protease resistance. Importantly, non-substrate analogs such as 1,3-propanediol and β-methylcholine failed to induce protection, underscoring the specificity of ligand-induced stabilization. These findings reveal a broadly conserved mechanism of substrate-induced conformational stabilization in GREs and B12-dependent radical enzymes and offer a scalable strategy for ligand identification with potential applications in enzyme engineering.
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Dec 2025
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I20-Scanning-X-ray spectroscopy (XAS/XES)
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Open Access
Abstract: We report the first experimental discovery of Hidden Satellites within the Kemission lines of manganese metal (Mn, ) with a total integrated statistical significance exceeding 270 (standard error), far beyond the discovery threshold. Experimental data were collected at the I20-Scanning beamline at the Diamond Light Source using our new eXtended-Range High-Energy-Resolution Fluorescence Detection (XR-HERFD) technique. The Hidden Satellites, embedded in the core emission structure, represent novel quantum many-body processes that evolve systematically as the incident photon energy increases. Principal Component Analysis (PCA) was applied to extract the major separable physical processes and validate the significance of the observed Hidden Satellites. The application of physical insight to the PCA method allowed us to isolate the satellites, and measure the evolutionary profile. Our paper reveals that the total intensity of shake-off satellites can reach as high as 20–25%. Although these are hidden, they are very significant. These results directly challenge the traditional treatment of the many-body reduction factor, , as a constant in the standard XAFS equation. Our findings demonstrate that this term must be modelled as an energy-dependent function, reflecting its variation with incident photon energy and highlighting its role in many-body interactions. This deeper understanding of fundamental atomic processes directly impacts relativistic quantum mechanics, in theory and application. Also, this develops the two most popular experimental techniques at synchrotrons: X-ray absorption and X-ray emission spectroscopy, responsible for some 12,000 papers per annum, and all applications of these techniques in chemistry, physics, and biology. It offers insights into the evolution of satellites and underscores the broader implications of hidden features in X-ray spectra.
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Dec 2025
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B18-Core EXAFS
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Diamond Proposal Number(s):
[29950]
Open Access
Abstract: The current research reports an inexpensive and effective method of synthesising δ bismuth oxide stable at room temperature by reacting monoclinic α-Bi2O3 and 5.25% sodium hypochlorite at 100 °C. The resultant product was washed, dried and characterized using optical imaging, microstructural analysis by scanning electron microscopy (SEM), thermographic assessment (TGA), X-ray diffraction (XRD), infrared spectroscopy (FT-IR), X-ray absorption fine structure (XAFS) and synchrotron XRD. The interaction with sodium hypochlorite resulted in a change in bismuth oxide microstructure and a colour change from yellow to dark brown/black. TGA showed a change in mass and colour with reversal at ~ 450 °C. Further analysis of bismuth oxide reacted with sodium hypochlorite revealed a stable δ-phase at room temperature with reformation of the α polymorph at 500 °C. The δ-bismuth oxide was modelled to show a defective fluorite structure similar to that proposed by Sillen. The current research is the first to report a cheap and effective method of stabilising the δ phase which is useful for a number of industrial processes such as an oxide ion conductor.
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Dec 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[26617]
Open Access
Abstract: TRF1 is a subunit of the shelterin complex that binds to and protects the linear ends of chromosomes known as telomeres. Both genetic deletion and chemical inhibition of TRF1 have been shown to block the growth of lung carcinoma, glioblastoma, and renal cell carcinoma in mice without affecting mouse survival or tissue function, making TRF1 a potential therapeutic target in cancer1,2,3. Here, we report the discovery of a series of fragment hits that bind at the interface between the TRFH domain of TRF1 (TRF1TRFH) and a peptide of TIN2 (TIN2TBM), an interaction essential for the recruitment of TRF1 to shelterin, using X-ray crystallography (XChem) and ligand-observed NMR (LO-NMR) fragment screening. We discovered a first-in-class inhibitor of the TRF1:TIN2 interaction (compound 40) that binds to TRF1TRFH with a KD of 29 µM (95% CI: 20–41 µM), displaces a TIN2 probe with an IC50 of 67 µM (95% CI: 10–120 µM), and expels TRF1 from purified shelterin. Aided by a novel crystal system of TRF1TRFH, we characterised fragments binding in a hotspot at the TRF1:TIN2 interface; these will serve as a starting point for the structure-guided development of potent inhibitors of TRF1 protein:protein interactions to disrupt shelterin complex assembly.
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Nov 2025
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B23-Circular Dichroism
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Open Access
Abstract: The emergence of multidrug-resistant (MDR) bacterial pathogens is an alarming global health threat that demands new therapeutic strategies beyond conventional antibiotics. Here, we present a rationally designed antimicrobial peptide (AMP) derived from mammalian cathelicidins and defensins that selectively targets bacterial membranes with low cytotoxicity toward mammalian cells. Circular dichroism spectroscopy revealed that the peptide adopts an α-helical conformation upon membrane interaction, a key feature of its mechanism. Surface plasmon resonance and isothermal titration calorimetry demonstrated high-affinity and selective binding to bacterial lipid membranes. Functionally, the peptide was strongly bactericidal against clinical MDR Escherichia coli (E. coli) and clinically important ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.). Compared with the parent peptide LL-37, our AMP exhibited lower minimum inhibitory concentrations (MICs) and faster bactericidal kinetics across both Gram-negative and Gram-positive strains. Calcein leakage assays, showing effective membrane disruption. Importantly, cytotoxicity experiments with human epithelial (Caco-2) and immune (THP-1) cells indicated low cytotoxicity at concentrations exceeding bactericidal levels, supporting a favorable therapeutic window. ELISA quantifications of cytokines (IL-6, TNF-α) further suggested immunomodulatory effects at bactericidal concentrations. Transcriptomic profiling of E. coli treated with sub-lethal concentrations of the peptide exhibited upregulation of bacterial stress response pathways and downregulation of vital metabolic processes, reflecting the complex antimicrobial action of the peptide. Collectively, these findings highlight this LL-37-derived AMP as a promising candidate for treating MDR bacterial infections caused by E. coli and ESKAPE pathogens and for guiding the development of next-generation antimicrobial agents.
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Oct 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[35585]
Open Access
Abstract: Peptide-based nanogels (NGs) represent a cutting-edge class of nanoscale drug delivery systems. Due to their structural properties, NGs platforms can encapsulate and protect therapeutic agents (e.g. peptides, proteins, and nucleic acids), while allowing for controlled and stimuli-responsive release. These pharmacokinetic and pharmacodynamic features can be specifically tuned by including peptide functional elements as NG components. This study explores the formulation, decoration strategies, and structural properties of NGs derived from mixed hydrogel (HG) matrices of Fmoc-diphenylalanine (Fmoc-FF) with cationic amphiphilic peptides (CAPs). CAPs, composed by cationic hexapeptide (GK)3 sequence decorated at its N-terminus with alkyl chain, were found able to confer a net positive charge to Fmoc-FF NGs. Fmoc-FF/C16-(GK)3 and Fmoc-FF/C18-(GK)3 NGs were obtained using polysorbate 80 (TWEEN 80) and sorbitane monooleate 80 (SPAN 80) as colloidal stabilizing surfactants and characterized in terms of size, secondary structure, superficial charge and shelf stability by Dynamic Light Scattering (DLS), Circular Dichroism (CD), Fourier Transform Infrared (FT-IR) and Small-Angle X-ray Scattering (SAXS) technique. Different formulative routes were used and mutually compared to encapsulate or adsorb AlexaFluor 430 (succinimidyl ester), used as model of an anionic, pharmaceutical agent. In vitro experiments demonstrated good cytocompatibility of these systems and the release of AlexaFluor 430 was also evaluated.
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Oct 2025
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Jingjing
Zhao
,
Chen
Huang
,
Ali
Mostaed
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Amirafshar
Moshtaghpour
,
James M.
Parkhurst
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Ivan
Lobato
,
Marcus
Gallagher-Jones
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Judy S.
Kim
,
Mark
Boyce
,
David
Stuart
,
Elena A.
Andreeva
,
Jacques-Philippe
Colletier
,
Angus I.
Kirkland
Open Access
Abstract: Exit wavefunction reconstruction is important in transmission electron microscopy for structural studies. We describe electron Fourier ptychography and its application to phase reconstruction of both radiation-resistant and beam-sensitive materials. We demonstrate that the phase of the exit wave can be reconstructed to high resolution using a modified iterative phase retrieval algorithm from data collected in an alternative optical geometry. This method achieves a spatial resolution of 0.63 nm at a fluence of 4.5 × 102 e−/nm2, as validated on Cry11Aa protein crystals under cryogenic conditions. Notably, this method requires no instrumental modifications, is straightforward to implement, and can be seamlessly integrated with existing data collection software, providing a broadly accessible alternative approach for structural studies.
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Oct 2025
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[38716]
Open Access
Abstract: Filamentous microbial biosignatures associated with iron sulfides are among the prime targets in early life studies, but their formation and preservation are insufficiently understood. Here, we experimentally evaluated the taphonomy of filamentous sulfur-oxidizing bacteria exposed to iron–sulfur–rich conditions and high temperatures (≤ 80 °C), mimicking burial diagenesis and/or hydrothermal alteration. The addition of ferrihydrite and sulfide at 22 °C resulted in a near-instantaneous formation of iron sulfides. Heating to 80 °C for 2–6 weeks resulted in the formation of polysulfides and magnetic Fe- and/or S-containing minerals, with low pyritization (~ 11%). Notably, Fe–S mineral formation was only loosely associated with the filaments. However, intracellular elemental sulfur released from the sulfur-oxidizing bacteria re-precipitated extracellularly, coating individual filaments, possibly promoting the formation of pyritic crusts during later diagenetic stages. Taken together, our study revealed that biosignatures in filamentous sulfur mats might be preserved in a variety of environments, including hydrothermal systems on and beyond the Earth.
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Oct 2025
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B07-B1-Versatile Soft X-ray beamline: High Throughput ES1
B07-C-Versatile Soft X-ray beamline: Ambient Pressure XPS and NEXAFS
Optics
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Wai Jue
Tan
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Arindam
Majhi
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Wadwan
Singhapong
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Andrew C.
Walters
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Matthijs A.
Van Spronsen
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Georg
Held
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Burcu
Karagoz
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David C.
Grinter
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Pilar
Ferrer
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Guru
Venkat
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Qiushi
Huang
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Zhe
Zhang
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Zhanshan
Wang
,
Patrick Yuheng
Wang
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Andrey
Sokolov
,
Hongchang
Wang
,
Kawal
Sawhney
Open Access
Abstract: X-ray Photoelectron Spectroscopy (XPS) is a powerful tool for probing the chemical and electronic states of materials with elemental specificity and surface sensitivity. However, its application in the tender X-ray range (1–5 keV) for synchrotron radiation has remained limited due to the limited choice of optics capable of maintaining high reflectivity and efficiency in this energy window. To address this, multilayer (ML) grating structures have become increasingly popular, offering significantly higher efficiency than SL coatings in the tender X-ray region. This paper presents the development of ML laminar gratings optimised for enhancing efficiency in the tender X-ray range, and capable of retaining performance under intense X-ray exposure in the oxygen partial pressure of 10 mbar. The ML coating quality was verified through X-ray reflectivity (XRR), XPS and near-edge X-ray absorption fine structures (NEXAFS) measurements, while the performance of the grating was validated through beamline flux transmission and XPS measurements. The MLLG demonstrated 22 higher intensity in flux and XPS, significantly improving the signal-to-noise ratio. Most importantly, the MLLGs outperformed traditional designs by offering improved spectral resolution while maintaining measurement capability at varying values without compromising the intensity. Furthermore, we demonstrated that the incorporation of nitrogen during deposition further enhances flux transmission.
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Oct 2025
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I11-High Resolution Powder Diffraction
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Diamond Proposal Number(s):
[40562]
Open Access
Abstract: The paper presents the study of phase and structural behavior of pseudoternary compound (Ga1-x-yInxAly)2O3 with focus on the compositional cross-section where the x/y ratio is a fixed at 0.31/0.69. This specific ratio ensures that the average cation radius, equal to that of Ga3+ ions, remains unchanged. Through the combination of experimental XRD studies and DFT calculations, the stability region of the monoclinic phase within the Ga2O3–Al2O3–In2O3 ternary system was established. Detailed analysis of the crystal lattice parameters and unit cell volume of the monoclinic structure was carried out across a wide range of compositions. An empirical relationship was derived linking the monoclinic lattice parameters to the average ionic radius of the cations (Ga3+, Al3+, In3+) enabling prediction of lattice parameters in monoclinic (Ga1-x-yInxAly)2O3 solely from chemical composition. The experimental crystal structure studies and the electronic structure calculations suggest that in the monoclinic (Ga1-x-yInxAly)2O3 structure the tetrahedral positions of Ga1 atoms are preferentially occupied by Ga3+ and Al3+ cations, while the octahedral Ga2 sites accommodate a mixture of Ga3+, Al3+ and In3+ cations. Additionally, the presence of unidentified phase(s) was confirmed in the central region of the Ga2O3–Al2O3–In2O3 triangle. Comparison of the calculated optical absorption spectra and the Tauc-plots derived from the diffuse reflectance spectra indicate that the monoclinic (Ga1−x−yInxAly)2O3 compounds have a direct band gap.
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Oct 2025
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