Protein crystallographers wish to have an accurate and fast X-ray detector in experimental states including their laboratories or synchrotron facilities. Imaging-plate and CCD detectors are available for this purpose with its effective dynamic-range and sensitivities rather than a classical two-dimensional detector, X-ray film. Such 2-D detectors bring a newly research field on the structural biology in terms of supra-molecular assembly, high precision analysis and high-throughput determination. Also more-fast detector will tempt us into a new protein diffractometry.

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Our study of electron crystallography or convergent beam electron diffraction (CBED) and of electron spectroscopy or electron energy-loss spectroscopy (EELS), which were done from the time when we introduced the energy filtering technique until my retirement, is described together with the important results. Crystal structure refinement was successfully carried out by fitting the energy filtered CBED pattern with the theoretically calculated pattern. A case study of the determination of a charge density distribution is shown. A high energy-resolution EELS instrument (< 0.1 eV) was developed for the study of the electronic structure or the structure of the conduction band. A case study of a detailed band structure is shown with a theoretical calculation. A clean application to reveal the optical properties of fullerenes is shown. A recent development of the wavelength dispersive spectroscopy attachment is mentioned. A small reminiscence on my research life is given.

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Several X-ray optical elements based on multilayer have been recently developed and have come onto the market. Its reflectivity is above 70 % for CuKα radiation. As these elements are artificially synthesized by means of spattering technique, it is possible to control the spacing of multilayer with its location and also possible to bend it along a geometrical curve. Thus, those elements have many applications to X-ray scattering and diffraction as various concave mirrors became available for X-rays. Next generation X-ray source, which should be used in conjunction with the multi-layer optics, is discussed in the relation with their characteristics.

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Endopolygalacturonases are involved in the degradation of pectin by hydrolyzing the α-1, 4 glycosidic bonds. Crystal structure of endopolygalacturonase I from Stereum purpureum was solved at atomic (0.96 Å) resolution. The enzyme folds into a right-handed parallel β-helix with 10 complete turns. The crystal structures of its binary complex with one D-galacturonate a and its ternary complex with two D-galacturonates were also determined at 1.0 and 1.15 Å resolutions, respectively. The active site architecture of the complexes provides insight into the mode of substrate binding. These structures reveal that Asp 173 is the general-acid catalyst and, Asp 153 or Asp 174 is the general-base catalyst.

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X-ray diffraction has been used to reveal precise kinetic data for the single-crystal-to-single-crystal photodimerization reaction of 2-benzyl-5-benzylidenecyclopentanone. The reaction obeys pseud-first-order kinetics. The reaction rate dependence on temperature is changed markedly at a threshold temperature of 200 K. This phenomenon is related to a change in the intermolecular distance of the two reactive olefin groups in association with temperature. A dimeric pair formation of the monomer molecules on crystallization is suggested to explain the structural similarity of the monomer and dimer crystals. Intermolecular interaction energy of the pair structure has been estimated by using a precise ab initio molecular orbital calculation.

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In order to investigate dioxygen-activation with dimetal center, we have introduced a teterahedral distortion into the Cu^III₂ (μ-O) ₂ core by using (-) -Sparteine (Sp) . X-ray studies of these copper complexes with Sp revealed that these copper centers have tetrahedrally distorted structures, ranging over the oxidation states, and the monomer and bridged dimer forms, due to the steric requirements of the ligand. The Cu^III₂ (μ-O) ₂ complex with Sp exhibited unique spectroscopic properties due to its structural distortion. On the basis of theoretical studies on these systems, the relationships between the physical properties and tetrahedrally-distorted structures have been discussed.

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Autocrine motility factor (AMF), a tumor-secreted cytokine, stimulates cell migration in vitro and metastasis in vivo. AMF is genetically identical to the extracellular cytokines neuroleukin (NLK) and maturation factor (MF) and, interestingly, to the intracellular enzyme phosphoglucose isomerase (PGI) . The crystal structures of the inhibitor-free open form and the inhibitor-bound closed form of human AMF have been determined at 1.9 Å and 2.4 Å resolution, respectively. Upon inhibitor binding, local conformation changes occur around the inhibitor-binding site. The inhibitor-bound structure shows that the location of the inhibitor (of cytokine activity) binding site of human AMF is very similar to those of the inhibitor (of enzymatic activity) binding sites of PGIs. The present study clearly shows that there is structural overlap of the regions responsible for the enzymatic and cytokine functions of AMF/PGI and suggests two scenarios for the inhibition mechanism of cytokine activity of AMF by the carbohydrate phosphate.

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