Search Results (41)

Serial crystallography (SX) determines the crystal structures of target molecules at room temperature with minimal radiation damage. During SX data collection, the stable delivery of many microcrystals to the X-ray interaction point is crucial for efficient sample consumption and effective beamtime usage. Most microcrystal delivery techniques for SX require sophisticated devices or specialized techniques, which can be challenging for data collection. This review introduces a straightforward method that delivers microcrystal samples in SX experiments using a commercially available syringe and syringe pump. This method does not require specialized skills for sample delivery and can be tested in the laboratory prior to SX data collection at the beamline. Advantages and disadvantages of this method are also discussed, along with various application cases. This straightforward sample delivery approach is concluded to facilitate efficient SX data collection. Full article

Serial crystallography (SX) enables the determination of biologically relevant structures at room temperature while minimizing radiation damage. During SX experiments, the beam center on diffraction images can shift due to X-ray beam movements or detector displacement. Consequently, the geometry file for the beam center is optimized; however, the effects of deviations from the optimal position on data processing efficiency remain unclear. This study examines how changes in the beam center influence data quality by analyzing the indexing efficiency and structure refinement of lysozyme and glucose isomerase datasets, considering shifts in the beam center parameter. The results revealed that as the beam center deviated farther from its optimal position, the indexing efficiency declined, with the extent of the effect varying significantly across indexing algorithms. XDS and MOSFLM algorithms maintained high indexing efficiencies (>90%) for shifts of ≤4 pixels (688 μm) and ≤2 pixels (344 μm), respectively, compared to data processed at the optimized beam center. Conversely, the DirAx and XGANDALF algorithms exhibited indexing efficiencies below 90% for a two-pixel shift in the beam center. These findings enhance our understanding of how beam center shifts affect SX data processing and provide valuable insights for developing effective data processing strategies. Full article

Lysozyme plays a crucial role in the natural immune system, protecting against invading bacteria or viruses. The room-temperature (RT) structure of lysozymes is important for understanding accurate structural information compared to the crystal structure determined at cryogenic temperature. Several RT structures of lysozymes are determined by serial crystallography, but their temperature-dependent structural properties are not fully elucidated. To better understand the temperature-dependent structural change, the RT and cryogenic temperature structures of hen egg white lysozyme (HEWL) were determined by serial synchrotron crystallography (SSX) and macromolecular crystallography (MX), respectively. Structural comparisons of HEWL^RT and HEWL^Cryo showed that the positions of the loops above the substrate-binding cleft of HEWL differed. The width of the substrate-binding cleft between the α- and β-domains of HEWL^RT was wider than that of HEWL^Cryo. The distance between the two catalytic residues Glu53 and Asp70 and their interaction with neighbor residues and water molecules showed the distant between HEWL^RT and HEWL^Cryo. Due to temperature, the subtle movements of the active site and substrate-binding cleft of HEWL led to different docking results for N-acetylglucosamine and N,N′,N″-triacetylchitotriose. These results will provide useful information to more accurately understand the molecular function of HEWL and insights into the temperature effects for ligand design. Full article

The voltage-dependent anion channel (VDAC) is a crucial mitochondrial protein that facilitates ion and metabolite exchange between mitochondria and the cytosol. Initially characterized over three decades ago, the structure of VDAC-1 was resolved in 2008, revealing a novel β-barrel protein architecture. This study presents the first room-temperature crystal structure of mouse VDAC-1 (mVDAC-1), which is a significant step toward understanding the channel’s gating mechanism. The new structure, obtained at a 3.3 Å resolution, demonstrates notable differences from the previously determined cryogenic structure, particularly in the loop regions, which may be critical for the transition between the ‘open’ and ‘closed’ states of VDAC-1. Comparative analysis of the root-mean-square deviation (R.M.S.D.) and B-factors between the cryogenic and room-temperature structures suggests that these conformational differences, although subtle, are important for VDAC’s functional transitions. The application of electric field-stimulated X-ray crystallography (EF-X) is proposed as a future direction to resolve the ‘closed’ state of VDAC-1 by inducing voltage-driven conformational changes in order to elucidate the dynamic gating mechanism of VDAC-1. Our findings have profound implications for understanding the molecular basis of VDAC’s role in mitochondrial function and its regulation under physiological conditions. Full article

A new coordination polymer {[Cd(C₁₂H₁₃O₅)₂(4,4′-bpy)(H₂O)₂]}_n (Cd-Tmca-bpy) was constructed with trans-2,3,4-Trimethoxycinnamic acid (HTmca) and 4,4′-Bipyridine (4,4′-bpy) ligands. This complex was structurally characterized on the basis of elemental analysis, infrared (IR) spectroscopy, powder X-ray diffraction and thermogravimetric analyses. X-ray crystallography revealed that the complex was monoclinic, space group C2/c. The Cd(II) ion in the complex was six coordinated, adopting an octahedron geometry. The neighboring Cd(II) ions linked linear ligand 4,4′-bpy molecules to form an infinite 1D chain. The 1D chain was further interlinked by O–H···O and C–H···O hydrogen bonds, resulting in a 3-D supramolecular framework. Meanwhile, the photoluminescence spectrum of the Cd(II) complex at room temperature exhibited an emission maximum at 475 nm. Using the time-dependent density functional theory (TD-DFT) method, the electronic absorption spectra of the Cd(II) complex was predicted. A good agreement was achieved between the predicted spectra and the experimental data. Bioactivity studies showed that the complex exhibited significant inhibition halos against Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus). Full article

Cu₂(ZnSn)(S)₄ (copper, zinc, tin, and sulfide (CZTS)) provides possible advantages over CuInGaSe2 for thin-film photovoltaic devices because it has a higher band gap. Preparing CZTS by electrodeposition because of its high productivity and lower processing costs, electroplating is appealing. Recently published studies reported that the electrodeposition process of CZTS still faces significant obstacles, such as the sulfur atomic ratio (about half of the whole alloy), deposits’ adhesion, film quality, and optical properties. This work introduces an improved bath that facilitates the direct electroplating of CZTS from one processing step. The precursors used were significantly more diluted than the typical baths mentioned in the last few years. An extensive analysis of the electrochemical behavior at various rotation speeds is presented at room temperature (~22 °C). The deposited alloy’s composition and adherence to the molybdenum back contact are examined with agitation. The annealing process was carried out in an environment containing sulfur, and the metal was not added at this stage. The ultimate sulfur composition was adjusted to 50.2%, about the desired atomic ratio. The compound’s final composition was investigated using the Energy-Dispersive X-ray Spectroscopy technique. Finally, X-ray diffraction analysis was applied to analyze CZTS crystallography and to measure thickness. 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

Enzymes are crucial in metabolic processes, and their dysfunction can lead to severe metabolic disorders. Structural biology, particularly X-ray crystallography, has advanced our understanding of these diseases by providing 3D structures of pathological enzymes. However, traditional X-ray crystallography faces limitations, such as difficulties in obtaining suitable protein crystals and studying protein dynamics. X-ray free-electron lasers (XFELs) have revolutionized this field with their bright and brief X-ray pulses, providing high-resolution structures of radiation-sensitive and hard-to-crystallize proteins. XFELs also enable the study of protein dynamics through room temperature structures and time-resolved serial femtosecond crystallography, offering comprehensive insights into the molecular mechanisms of metabolic diseases. Understanding these dynamics is vital for developing effective therapies. This review highlights the contributions of protein dynamics studies using XFELs and synchrotrons to metabolic disorder research and their application in designing better therapies. It also discusses G protein-coupled receptors (GPCRs), which, though not enzymes, play key roles in regulating physiological systems and are implicated in many metabolic disorders. Full article

The reaction between 5-acetylbarbituric acid and 4-dimethylthiosemicarbazide or 4-hexamethyleneiminyl thiosemicarbazide produces 5-acetylbarbituric-4-dimethylthiosemicarbazone (H₂AcbDM) and 5-acetylbarbituric-4N-hexamethyleneiminyl thiosemicarbazone (H₂Acbhexim). Eight new complexes with different copper(II) salts have been prepared and characterized using elemental analysis, molar conductance, UV–Vis, ESI-HRMS, FT-IR, magnetic moment, EPR, and cyclic voltammetry. In addition, three-dimensional molecular structures of [Cu(HAcbDM)(H₂O)₂](NO₃)·H₂O (3a), [Cu(HAcbDM)(H₂O)₂]ClO₄ (4), and [Cu(HAcbHexim)Cl] (6) were determined by single crystal X-ray crystallography, and an analysis of their supramolecular structure was carried out. The H-bonded assemblies were further studied energetically using DFT calculations and MEP surface and QTAIM analyses. In these complexes, the thiosemicarbazone coordinates to the metal ion in an ONS-tridentate manner, in the O-enolate/S-thione form. The electrochemical behavior of the thiosemicarbazones and their copper(II) complexes has been investigated at room temperature using the cyclic voltammetry technique in DMFA. The Cu(II)/Cu(I) redox system was found to be consistent with the quasi-reversible diffusion-controlled process. Full article

Manganese catalysts that activate hydrogen peroxide carry out several different hydrocarbon oxidation reactions with high stereoselectivity. The commonly proposed mechanism for these reactions involves a key manganese(III)-hydroperoxo intermediate, which decays via O–O bond heterolysis to generate a Mn(V)–oxo species that institutes substrate oxidation. Due to the scarcity of characterized Mn^III–hydroperoxo complexes, Mn^III–alkylperoxo complexes are employed to understand factors that affect the mechanism of the O–O cleavage. Herein, we report a series of novel complexes, including two room-temperature-stable Mn^III–alkylperoxo species, supported by a new amide-containing pentadentate ligand (^6Medpaq^5NO2). We use a combination of spectroscopic methods and density functional theory computations to probe the effects of the electronic changes in the ligand sphere trans to the hydroxo and alkylperoxo units to thermal stability and reactivity. The structural characterizations for both Mn^II(OTf)(^6Medpaq^5NO2) and [Mn^III(OH)(^6Medpaq^5NO2)](OTf) were obtained via single-crystal X-ray crystallography. A perturbation to the ligand sphere allowed for a marked increase in reactivity towards an organic substrate, a modest change in the distribution of the O–O cleavage products from homolytic and heterolytic pathways, and little change in thermal stability. Full article

The carboxylation of unsaturated amine and alcohol compounds, including 4-benzylamino-1-phenyl-1-butyne (homopropargylamine), 2-butyne-1-ol (propargylic alcohol), and 2,3-butadiene-1-ol (allenylmethyl alcohol), using the hydroxidogold(I) complex, AuOH(IPr) [IPr = 1,3-bis(2,6-diisopropylphenyl)-imidazol-2-ylidene], produces corresponding alkenylgold(I) complexes with a cyclic urethane or carbonate framework in high yields. The reaction takes place in aprotic THF at room temperature under the atmospheric pressure of CO₂ in the absence of base additives. The products were characterized by NMR spectroscopy, elemental analysis, and X-ray crystallography. The functionalized alkenyl complexes prepared from the alkynes can be protonated by treatment with an equimolar amount of acetic acid to afford five- or six-membered carboxylation products, whereas the related alkenyl complex derived from allenylmethyl alcohol decomposed to recover the starting allene via ring-opening decarboxylation. Full article

Temperature directly influences the function and structure of proteins. Crystal structures determined at room temperature offer more biologically relevant structural information regarding flexibility, rigidity, and thermal motion than those determined by conventional cryocrystallography. Crystal structures can be determined at room temperature using conventional macromolecular crystallography (MX) or serial crystallography (SX) techniques. Among these, MX may theoretically be affected by radiation damage or X-ray heating, potentially resulting in differences between the room temperature structures determined by MX and SX, but this has not been fully elucidated. In this study, the room temperature structure of xylanase GH11 from Thermoanaerobacterium saccharolyticum was determined by MX (RT-TsaGH11-MX). The RT-TsaGH11-MX exhibited both the open and closed conformations of the substrate-binding cleft within the β-sandwich fold. The RT-TsaGH11-MX showed distinct structural changes and molecular flexibility when compared with the RT-TsaGH11 determined via serial synchrotron crystallography. The notable molecular conformation and flexibility of the RT-TsaGH11-MX may be induced by radiation damage and X-ray heating. These findings will broaden our understanding of the potential limitations of room temperature structures determined by MX. Full article

To resolve photons hungry for weak diffraction samples by the crystallographic method, a double-multilayer monochromator (DMM) was employed on an undulator beamline (BL17UM) at the Shanghai Synchrotron Radiation Facility (SSRF) to provide a focused sub-micron beam with high brightness for macromolecular crystallography experiments. High-quality crystallographic datasets from model protein crystal samples were collected and processed by an existing crystallographic program for structure solution and refinement. The data quality was compared with datasets from a normal silicon crystal monochromator to evaluate the bandwidth of the DMM effect on these crystallographic data. This experiment demonstrates that multilayer optics on an undulator beamline may play a valuable role in satisfying the demands of structure-related research, which requires novel methods. Full article

Serial crystallography (SX) enables the determination of the structure of macromolecules or small molecules with minimal radiation damage. In particular, biomolecule structures determined using the SX technique have the advantage of providing room-temperature crystal structures with high biological relevance. The SX technique requires numerous crystals to be collected to complete three-dimensional structural information. To minimize crystal sample consumption, we introduced SX data collection with fixed-target (FT) pink-beam serial synchrotron crystallography (SSX) at the 1C beamline of Pohang Light Source II. A new sample holder consisting of a magnetic frame with a nylon mesh was developed for easy sample handling. The FT-pink-SSX diffraction data were collected by continuously scanning X-rays using a stepping motor. The room-temperature structures of glucose isomerase and lysozyme were successfully determined at a resolution of 1.7 and 2.2 Å, respectively. The use of pink-beam FT-SSX in experimental applications and data acquisition for large beam sizes is discussed. Our results provide useful information for future pink-beam SSX and SX data collection using large X-ray beams. Full article

Serial crystallography is a rapidly developing method for the determination of the structure of biomolecules at room temperature at near-atomic resolution from an ensemble of small crystals. Numerous advances in detectors, data analysis pipelines, sample delivery methods, and crystallization protocols expand the scope of structural biology to understand the fundamental processes that take place in living cells. Many experimental strategies for serial crystallography are in use, depending on the type and sizes of the crystals or other needs of the experiment. Such strategies should ideally minimize the wastage of samples or beamtime without compromising experimental goals. This paper proposes a way to optimize beamtime utilization in serial crystallography experiments that use fixed-target sample delivery methods, such as chips. The strategy involves two key steps. Firstly, a fast raster scan of the chip is performed to determine the positions of the crystals based on their diffraction. Subsequently, a rotational series is collected at each identified crystal position, covering a narrow range of chip orientations. This approach enables the exclusion of empty positions during data acquisition, resulting in significant savings in beam time utilization and a reduced volume of measured data. Full article