Search Results (44)

Serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) enables the determination of room-temperature structures of biological macromolecules without radiation damage. The accuracy of detector geometry parameters, including the crystal-to-detector distance (CTDD), is critical for reliable data processing. In SFX experiments, the CTDD may shift during data collection due to changes in the experimental setup or installation of the sample delivery system. Such CTDD variations can affect the quality of SFX datasets; however, their impact has not been fully elucidated in the context of SFX data processing. In this study, we investigated the influence of CTDD variations on SFX datasets collected at Pohang Accelerator Laboratory X-ray Free Electron Laser (PAL-XFEL) with thermolysin, lysozyme, and glucose isomerase crystals processed by four indexing algorithms. At the optimized CTDD, the distribution of unit cell parameters exhibited a Gaussian pattern; however, it became distorted as the CTDD deviated further from the optimal value. Data analysis indicated that the CTDD tolerance for successful data processing and structure determination was approximately ±3–5 mm from the optimized CTDD. These findings provide insight into indexing behavior in SFX data processing at PAL-XFEL and offer practical guidance for improving data processing efficiency. Full article

The development of serial crystallography (SX), including serial synchrotron crystallography (SSX) at synchrotron sources and serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs), has facilitated the collection of high-resolution diffraction data from micron-sized crystals, providing unique insights into the structures and dynamics of biomolecules at room temperature. Standard samples are essential for the commissioning of new XFEL instruments and the validation of experimental setups. In this review, we summarize currently used standard proteins and describe representative microcrystal preparation workflows for four widely adopted models, lysozyme, myoglobin, iq-mEmerald, and photoactive yellow protein (PYP), drawing on established methodologies and accumulated experience from their applications at the European XFEL. By consolidating existing knowledge and integrating protocols that have been systematically refined and optimized through our experimental efforts, this review aims to provide practical guidance for the serial crystallography community, thereby enhancing reproducibility and ensuring consistent experimental performance across facilities. Full article

Myoglobin (Mb), a heme-containing protein, plays crucial roles in storing and transporting oxygen in muscle cells. Various Mb structures have been extensively determined using conventional cryogenic crystallography, providing valuable information for understanding the molecular mechanisms of the protein. However, this approach has limitations attributable to cryogenic temperatures and radiation damage. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers is an emerging technique that enables the determination of biologically relevant room-temperature structures without causing radiation damage. In this study, we assessed the crystallization, collection, and processing of SFX diffraction data of Mb from equine skeletal muscle. Needle- and needle cluster-shaped Mb crystals were obtained using the microbatch method. Fixed-target SFX data collection was performed at the Pohang Accelerator Laboratory X-ray Free Electron Laser, yielding 1389 indexed diffraction patterns. The phase problem was solved by molecular replacement. The preliminary Mb structure determined at 2.3-Å resolution in this study exhibited subtle structural differences in the heme environment compared with previously reported Mb structures determined by SFX. These results both confirm the feasibility of myoglobin SFX experiments and establish a foundation for future time-resolved studies aiming to visualize ligand binding and oxygen transport. Full article

Propranolol is a non-selective β-adrenergic receptor antagonist widely used in cardiovascular and neurological therapy. Its naphthalene-based structure contributes to its high lipophilicityand central nervous system penetration. Clinically, propranolol is indicated for hypertension, arrhythmias, anxiety, migraine, and other conditions. It undergoes extensive hepatic metabolism via cytochrome P450 enzymes, notably CYP2D6, with a significant first-pass effect limiting oral bioavailability. This review integrates pharmacological profiling with crystallographic analysis to explore propranolol’s molecular interactions and therapeutic versatility. High-resolution crystal structures of the human β₂-adrenergic receptor (hβ₂-AR), particularly PDB ID: 6PS5 obtained via serial femtosecond crystallography (SFX), reveal key binding determinants responsible for receptor affinity and antagonism. Comparative structural analysis with other β-blockers—alprenolol, timolol, and carvedilol—highlights how variations in aromatic and heterocyclic frameworks influence pharmacokinetics and receptor selectivity. Superimposition results (RMSD: 0.032 for propranolol–alprenolol, 0.078 for propranolol–carvedilol, and 1.078 for propranolol–timolol) quantitatively illustrate molecular similarity and divergence. The enantioselective behavior of propranolol is also discussed, with the S-enantiomer showing greater receptor affinity and pharmacological potency than the R-form. Beyond canonical β-adrenergic targets, propranolol interacts with non-canonical proteins such as the cellulase enzyme Cel7A and lactoferrin, suggesting off-target effects and novel therapeutic potential. These findings underscore the importance of propranolol’s amphiphilic character, stereochemistry, and electrostatic properties in shaping its pharmacological profile. Overall, the integration of crystallographic data with pharmacological insights supports the rational design of next-generation β-adrenergic ligands with enhanced selectivity, bioavailability, and clinical efficacy. Full article

Neonicotinoid insecticides (NEOs), one of the most widely used pesticide classes worldwide, have raised concerns due to potential neurotoxic effects. Yet evidence on human exposure and health outcomes, particularly in preschool children, remains limited. In this study, 506 children aged 3–6 years from Shenzhen, China, were assessed. Neurobehavioral development was evaluated with the Strengths and Difficulties Questionnaire (SDQ), and urinary concentrations of 11 NEOs were measured, including imidacloprid (IMI), clothianidin (CLO), thiamethoxam (THM), dinotefuran (DNT), nitenpyram (NIT), sulfoxaflor (SFX), acetamiprid (ACE), thiacloprid (THD), flonicamid (FLO), 6-chloronicotinic acid (6-CINA), N-desmethyl-acetamiprid (NACE), and N-desmethyl-thiamethoxam (NTHM). Seven compounds showed high detection rates, including IMI (97.4%), CLO (100%), THM (100%), DNT (99.8%), NIT (99.8%), NACE (100%), and NTHM (99.8%). The mean urinary concentration was 234.145 μg/g creatinine, exceeding levels in earlier studies and indicating widespread exposure. IMI, NTHM, and NACE showed significant positive dose–response relationships with emotional symptoms, hyperactivity, and total difficulties and were major contributors in mixture models; sex-stratified analyses suggested effect modification for NTHM and NACE. These findings provide new epidemiological evidence to inform public health risk assessment and regulatory action on NEOs. Full article

For active control of impulsive noise, the conventional filtered-x least mean square (FxLMS) algorithm has poor noise reduction performance. To address this issue, this paper designs a robust cost function by embedding the cost function of the FxLMS algorithm into the framework of the adjustable Softsign function, thereby designing a robust Softsign-FxLMS (SFxLMS) algorithm for ANC systems. Furthermore, the parameter λ of the SFxLMS algorithm significantly influences its robustness and convergence speed. Therefore, a variable λ-parameter SFxLMS (VSFxLMS) algorithm is designed to improve the performance of the ANC system. Simulation studies indicate that the proposed SFxLMS algorithm and VSFxLMS algorithm exhibit stronger robustness, faster convergence rates, and better tracking performance compared to several robust FxLMS algorithms. Moreover, the symmetric properties of the proposed Softsign function contribute to balanced error suppression in both positive and negative directions, enhancing the robustness and stability of the ANC system under asymmetric impulsive noise conditions. Full article

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) enables the determination of biological and chemical structures without radiation damage. In SFX experiments, a sample delivery system is essential for delivering numerous crystals to the X-ray interaction point in a serial and stable manner. Among the various sample delivery methods, the fixed-target (FT) sample delivery system is straightforward and widely used for collecting SFX data due to its advantages of low sample consumption and reduced physical damage to crystals during data collection. Here, we review the development of the FT sample delivery system for SFX with the Pohang Accelerator Laboratory X-ray free-electron laser (PAL-XFEL). The specifications and operational conditions of the FT-SFX sample chamber are described. The design, specifications, and applications of the one- and two-dimensional FT sample holders developed for SFX with the PAL-XFEL are also detailed. Furthermore, the applications of each FT sample delivery system are discussed. This review not only provides valuable information on the FT system used in SFX experiments with the PAL-XFEL but also offers insights into the development of FT sample delivery systems. Full article

This work aims to verify whether the continuum mechanics assumption holds for the numerical simulation of a typical sample delivery system in serial femtosecond crystallography (SFX). Knudsen numbers were calculated based on the numerical simulation results of helium flow through the gas-focused liquid sheet nozzle into the vacuum chamber, representing the upper limit of Knudsen number for such systems. The analysed flow is considered steady, compressible, and laminar. The numerical results are mesh-independent, with a Grid Convergence Index significantly lower than 1% for global and local analysis. This study is based on an improved definition of the numerical Knudsen number: a combination of the cell Knudsen number and the physical Knudsen number. In the analysis, no-slip boundary and low-pressure boundary slip conditions are compared. No significant differences are observed. This study justifies using computational fluid dynamics (CFD) analysis for SFX sample delivery systems based on the assumption of continuum mechanics. Full article

Atopic dermatitis (AD) is characterized by a T-helper cell type 2 (Th2) inflammatory response leading to skin damage with erythema and edema. Comparative fecal sample analysis has uncovered a strong correlation between AD and Faecalibacterium prausnitzii strain A2-165, specifically associated with butyrate production. Therefore, understanding the functional mechanisms of crucial enzymes in the butyrate pathway, such as 3-hydroxybutyryl-CoA dehydrogenase of A2-165 (A2HBD), is imperative. Here, we have successfully elucidated the three-dimensional structure of A2HBD in complex with acetoacetyl-CoA and NAD⁺ at a resolution of 2.2Å using the PAL-11C beamline (third generation). Additionally, X-ray data of A2HBD in complex with acetoacetyl-CoA at a resolution of 1.9 Å were collected at PAL-XFEL (fourth generation) utilizing Serial Femtosecond Crystallography (SFX). The monomeric structure of A2HBD consists of two domains, N-terminal and C-terminal, with cofactor binding occurring at the N-terminal domain, while the C-terminal domain facilitates dimerization. Our findings elucidate the binding mode of NAD⁺ to A2HBD. Upon acetoacetyl-CoA binding, the crystal structure revealed a significant conformational change in the Clamp-roof domain (root-mean-square deviation of 2.202 Å). Notably, residue R143 plays a critical role in capturing the adenine phosphate ring, underlining its significance in substrate recognition and catalytic activity. The binding mode of acetoacetyl-CoA was also clarified, indicating its lower stability compared to NAD⁺. Furthermore, the conformational change of hydrophobic residues near the catalytic cavity upon substrate binding resulted in cavity shrinkage from an open to closed conformation. This study confirms the conformational changes of catalytic triads involved in the catalytic reaction and presents a proposed mechanism for substrate reduction based on structural observations. Full article

Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) has revolutionized classical X-ray diffraction experiments by utilizing ultra-short, intense, and coherent X-ray pulses. However, the SFX approach still requires thousands of nearly identical samples, leading to significant protein consumption. We propose utilizing Langmuir–Blodgett protein multilayers, which are characterized by long-range order, thermal stability, and the ability to induce protein crystallization, even in proteins that cannot be crystallized by conventional methods. This study aimed to combine the intrinsic properties of Langmuir–Blodgett multilayers with advanced XFEL techniques at the Linac Coherent Light Source. Since the macromolecule organization can be explored in nano or 2D crystals exploiting the properties of SFX–XFEL radiation that enable the capture of high-resolution diffraction images before radiation damage occurs, we propose Langmuir–Blodgett protein nanofilm technology as a novel approach for direct “on-chip” protein sample preparation. The present study extends previous investigations into Langmuir–Blodgett phycocyanin multilayer nanofilms using synchrotron radiation cryo-EM microscopy and second-order nonlinear imaging of chiral crystal (SONICC) experiments. We also examined the thermal stability of phycocyanin Langmuir–Blodgett multilayered films deposited on Si₃N₄ membranes to evaluate structural changes occurring at 150 °C compared with room temperature. Phycocyanin Langmuir–Blodgett films are worthy of investigation in view of their suitability for tissue engineering and other applications due to their thermal integrity and stability as the results of the present investigation reveal. Full article

In the field of perovskite optoelectronics, developing hole-transporting materials (HTMs) on the spiro[fluorene-9,9′-xanthene] (SFX) platform is one of the current research focuses. The SFX inherits the merits of spirobifluorene in terms of the configuration and property, but it is more easily derivatized and regulated by virtue of its binary structure. In this work, we design and synthesize four isomeric SFX-based HTMs, namely m-SFX-mF, p-SFX-mF, m-SFX-oF, and p-SFX-oF, through varying the positions of fluorination on the peripheral aniline units and their substitutions on the SFX core, and the optoelectronic performance of the resulting HTMs is evaluated in both perovskite solar cells (PSCs) and light-emitting diodes (PeLEDs) by the vacuum thermal evaporating hole-transporting layers (HTLs). The HTM p-SFX-oF exhibits an improved power conversion efficiency of 15.21% in an inverted PSC using CH₃NH₃PbI₃ as an absorber, benefiting from the deep HOMO level and good HTL/perovskite interface contact. Meanwhile, the HTM m-SFX-mF provides a maximum external quantum efficiency of 3.15% in CsPb(Br/Cl)₃-based PeLEDs, which is attributed to its perched HOMO level and shrunken band-gap for facilitating charge carrier injection and then exciton combination. Through elucidating the synergistic position effect of fluorination on aniline units and their substitutions on the SFX core, this work lays the foundation for developing low-cost and efficient HTMs in the future. Full article

Microcrystal delivery instruments are pivotal to performing serial femtosecond crystallography experiments at the XFEL facilities. We present a novel sample delivery technique based on a micro-tubing reeling system (MRS). Despite the tiny size of the micro-tubing, the MRS device has the advantage of operating without real-time position adjustment of the tube to match with the XFEL pulses. Moreover, the applicable repetition rate is more flexible than the previously reported chip-based one-dimensional fixed target system. Full article

Sulforaphane, the native but unstable form of SFX-01, is an antioxidant that activates the NRF2 and inhibits the NF-KB pathways to achieve its actions. Resolving the mechanism(s) by which SFX-01 serves to control the various osteoclastogenic stages may expose pathways that could be explored for therapeutic use. Here we seek to identify the stage of osteoclastogenesis targeted by SFX-01 and explore whether, like SFN, it exerts its actions via the NRF2 and NF-KB pathways. Osteoclasts generated from the bone marrow (BM) of mice were cultured with SFX-01 at different timepoints to examine each phase of osteoclastogenesis separately. This showed that SFX-01 exerted actions throughout the process of osteoclastogenesis, but had its largest effects in the early osteoclast precursor differentiation stage. Thus, treatment with SFX-01 for the duration of culture, for the initial 3 days differentiation or for as little as the first 24 h was sufficient for effective inhibition. This aligned with data suggesting that SFX-01 reduced DC-STAMP levels, osteoclast nuclear number and modified cytoskeletal architecture. Pharmacological regulation of the NRF2 pathways, via selective inhibitors/activators, supported the anti-osteoclastogenic roles of an SFX-01-mediated by NRF2 activation, as well as the need for tight NF-KB pathway regulation in osteoclast formation/function. Full article

X-ray free electron lasers deliver photon pulses that are bright enough to observe diffraction from extremely small crystals at a time scale that outruns their destruction. As crystals are continuously replaced, this technique is termed serial femtosecond crystallography (SFX). Due to its high pulse repetition rate, the European XFEL enables the collection of rich and extensive data sets, which are suited to study various scientific problems, including ultra-fast processes. The enormous data rate, data complexity, and the nature of the pixelized multimodular area detectors at the European XFEL pose severe challenges to users. To streamline the analysis of the SFX data, we developed the semiautomated pipeline EXtra-Xwiz around the established CrystFEL program suite, thereby processing diffraction patterns on detector frames into structure factors. Here we present EXtra-Xwiz, and we introduce its architecture and use by means of a tutorial. Future plans for its development and expansion are also discussed. Full article

For developing high-performance organic light-emitting diodes (OLEDs) with thermally activated delayed fluorescent (TADF) emitters, the diphenyltriazine (TRZ) unit was introduced onto the 2′- and 3′-positions of xanthene moiety of spiro[fluorene-9,9′-xanthene] (SFX) to construct n-type host molecules, namely 2′-TRZSFX and 3′-TRZSFX. The outward extension of the TRZ unit, induced by the meta-linkage, resulted in a higher planarity between the TRZ unit and xanthene moiety in the corresponding 3′-TRZSFX. Additionally, this extension led to a perched T₁ level, as well as a lower unoccupied molecular orbital (LUMO) level when compared with 2′-TRZSFX. Meanwhile, the 3′-TRZSFX molecules in the crystalline state presented coherent packing along with the interaction between TRZ units; the similar packing motif was spaced apart from xanthene moieties in the 2′-TRZSFX crystal. These endowed 3′-TRZSFX superior electron transport capacity in single-carrier devices relative to the 2′-TRZSFX-based device. Hence, the 3′-TRZSFX-based TADF–OLED showed remarkable electroluminescent (EL) performance under the operating luminance from turn-on to ca. 1000 cd·m⁻² with a maximum external quantum efficiency (EQE_max) of 23.0%, thanks to its matched LUMO level with 4CzIPN emitter and better electron transport capacity. Interestingly, the 2′-TRZSFX-based device, with an EQE_max of 18.8%, possessed relatively low roll-off and higher efficiency when the operating luminance exceeded 1000 cd·m⁻², which was attributed to the more balanced carrier transport under high operating voltage. These results were elucidated by the analysis of single-crystal structures and the measurements of single-carrier devices, combined with EL performance. The revealed position effect of the TRZ unit on xanthene moiety provides a more informed strategy to develop SFX-based hosts for highly efficient TADF–OLEDs. Full article