Publications by authors named "Naohiro Matsugaki"

Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength.

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MA026, a cyclic lipodepsipeptide, opens the tight junction (TJ) probably via binding to claudin-1. We reported that (1) TJ-opening activity is dependent on the amino acid sequence order at Glu10-Leu11; (2) an epimer at the C3 position of the -terminal acyl tail decreased the TJ-opening activity; and (3) the epimers D-Leu1/L-Gln6 and L-Leu1/D-Gln6 showed more potent TJ-opening activity than natural MA026, although no systematic structure-activity relationship (SAR) study was conducted. Here, we report the three-dimensional structure and systematic SAR study of MA026.

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While native SAD phasing is a promising method for next-generation macromolecular crystallography, it requires the collection of high-quality diffraction data using long-wavelength X-rays. The crystal itself and the noncrystalline medium around the crystal can cause background noise during long-wavelength X-ray data collection, hampering native SAD phasing. Optimizing the crystal size and shape or removing noncrystalline sample portions have thus been considered to be effective means of improving the data quality.

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Hybrid perovskites are among the most promising materials for optoelectronic applications. Their 2D crystalline form is even more interesting since the alternating inorganic and organic layers naturally forge a multiple quantum-well structure, leading to the formation of stable excitonic resonances. Nevertheless, a controlled modulation of the quantum well width, which is defined by the number of inorganic layers (n) between two organic ones, is not trivial and represents the main synthetic challenge in the field.

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crystallization is a developing technique to provide crystals for protein structure determination, particularly for proteins that are difficult to prepare by crystallization. This method has a key advantage: it requires neither a protein purification step nor a crystallization step. However, there is still no systematic strategy for improving the technique of crystallization because the process occurs spontaneously.

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Native single-wavelength anomalous dispersion (SAD) is an attractive experimental phasing technique as it exploits weak anomalous signals from intrinsic light scatterers ( < 20). The anomalous signal of sulfur in particular, is enhanced at long wavelengths, however the absorption of diffracted X-rays owing to the crystal, the sample support and air affects the recorded intensities. Thereby, the optimal measurable anomalous signals primarily depend on the counterplay of the absorption and the anomalous scattering factor at a given X-ray wavelength.

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The α--acetylgalactosaminidase from the probiotic bacterium (NagBb) belongs to the glycoside hydrolase family 129 and hydrolyzes the glycosidic bond of Tn-antigen (GalNAcα1-Ser/Thr). NagBb is involved in assimilation of -glycans on mucin glycoproteins by in the human gastrointestinal tract, but its catalytic mechanism has remained elusive because of a lack of sequence homology around putative catalytic residues and of other structural information. Here we report the X-ray crystal structure of NagBb, representing the first GH129 family structure, solved by the single-wavelength anomalous dispersion method based on sulfur atoms of the native protein.

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Orchestration of the multiple enzymes engaged in O-mannose glycan synthesis provides a matriglycan on α-dystroglycan (α-DG) which attracts extracellular matrix (ECM) proteins such as laminin. Aberrant O-mannosylation of α-DG leads to severe congenital muscular dystrophies due to detachment of ECM proteins from the basal membrane. Phosphorylation at C6-position of O-mannose catalyzed by protein O-mannosyl kinase (POMK) is a crucial step in the biosynthetic pathway of O-mannose glycan.

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The p24 family proteins form homo- and hetero-oligomeric complexes for efficient transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. It consists of four subfamilies (p24α, p24β, p24γ, and p24δ). p24γ2 plays crucial roles in the selective transport of glycosylphosphatidylinositol-anchored proteins.

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The protein crystallography beamline BL2S1, constructed at one of the 5 T superconducting bending-magnet ports of the Aichi synchrotron, is available to users associated with academic and industrial organizations. The beamline is mainly intended for use in X-ray diffraction measurements of single-crystals of macromolecules such as proteins and nucleic acids. Diffraction measurements for crystals of other materials are also possible, such as inorganic and organic compounds.

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Native SAD is an emerging phasing technique that uses the anomalous signal of native heavy atoms to obtain crystallographic phases. The method does not require specific sample preparation to add anomalous scatterers, as the light atoms contained in the native sample are used as marker atoms. The most abundant anomalous scatterer used for native SAD, which is present in almost all proteins, is sulfur.

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Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme-substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.

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The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals.

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BL-17A is a macromolecular crystallography beamline dedicated to diffraction experiments conducted using micro-crystals and structure determination studies using a lower energy X-ray beam. In these experiments, highly accurate diffraction intensity measurements are definitively important. Since this beamline was constructed, the beamline apparatus has been improved in several ways to enable the collection of accurate diffraction data.

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Photon Factory Automated Mounting system (PAM) protein crystal exchange systems are available at the following Photon Factory macromolecular beamlines: BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. The beamline AR-NE3A has been constructed for high-throughput macromolecular crystallography and is dedicated to structure-based drug design. The PAM liquid-nitrogen Dewar can store a maximum of three SSRL cassettes.

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The macromolecular crystallography (MX) beamline AR-NW12A is evolving from its original design of high-throughput crystallography to a multi-purpose end-station. Among the various options to be implemented, great efforts were made in making available high-pressure MX (HPMX) at the beamline. High-pressure molecular biophysics is a developing field that attracts the interest of a constantly growing scientific community.

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A subset of tumour necrosis factor receptor (TNFR) superfamily members contain death domains in their cytoplasmic tails. Death receptor 6 (DR6) is one such member and can trigger apoptosis upon the binding of a ligand by its cysteine-rich domains (CRDs). The crystal structure of the ectodomain (amino acids 1-348) of human death receptor 6 (DR6) encompassing the CRD region was phased using the anomalous signal from S atoms.

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A direct outcome of the exponential growth of macromolecular crystallography is the continuously increasing demand for synchrotron beam time, both from academic and industrial users. As more and more projects entail screening a profusion of sample crystals, fully automated procedures at every level of the experiments are being implemented at all synchrotron facilities. One of the major obstacles to achieving such automation lies in the sample recognition and centring in the X-ray beam.

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The generation of crystal lattice contacts by proteinaceous tags fused to target proteins is an attractive approach to aid in the crystallization of otherwise intractable proteins. Here, the use of green fluorescent protein (GFP) fusions for this purpose is demonstrated, using ubiquitin and the ubiquitin-binding motif (UBM) of Y-family polymerase ι as examples. The structure of the GFP-ubiquitin fusion protein revealed that the crystal lattice was formed by GFP moieties.

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The Targeted Protein Research Program (TPRP) started in 2007 as a sequel of the Protein 3000 Project which lasted from 2002 to 2007. In the new project, four cores, Protein Production, Structure Analysis, Control of Protein Functions with Compounds, and Informatics, have been established as focus of methodology developments critical for functional and structural studies by the target protein research teams. Within the "Analysis Core" synchrotron radiation plays a pivotal role providing X-ray beams for structural analyses of the target proteins.

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The X-ray crystal structure of natural commelinin is investigated. The results demonstrate that commelinin is a tetranuclear (4 Mg(2+)) metal complex, in which two Mg(2+) ions chelate to six anthocyanin molecules, while the other two Mg(2+) ions bind to six flavone molecules, stabilizing the commelinin complex, a new type of supramolecular complex.

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Sample-exchange robots that can exchange cryo-pins bearing protein crystals out of experimental hutches according to user instructions have been developed. The robots were designed based on the SAM (Stanford Synchrotron Research Laboratory automated mounting) system. In order to reduce the time required for the sample exchange, the single tongs of the SAM system were modified and a double-tongs system that can hold two cryo-pins at the same time was developed.

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Owing to recent advances in high-throughput technology in macromolecular crystallography beamlines, such as high-brilliant X-ray sources, high-speed readout detectors and robotics, the number of samples that can be examined in a single visit to the beamline has increased dramatically. In order to make these experiments more efficient, two functions, remote monitoring and diffraction image evaluation, have been implemented in the macromolecular crystallography beamlines at the Photon Factory (PF). Remote monitoring allows scientists to participate in the experiment by watching from their laboratories, without having to come to the beamline.

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BL-17A is a new structural biology beamline at the Photon Factory, Japan. The high-brilliance beam, derived from the new short-gap undulator (SGU#17), allows for unique protein crystallographic experiments such as data collection from microcrystals and structural determination using softer X-rays. However, microcrystal experiments require robust beam stability during data collection and minor fluctuations could not be ignored.

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