Journal of the Crystallographic Society of Japan
Volume 52 (1), 2010.

J. Cryst. Soc. Jpn., 52(1), 3-7 (2010).
Structural Studies on Membrane Proteins and Biological MacromolecularAssemblies in Japan
Affiliation: Institute Protein Research, Osaka University
Address: 3-2 Yamada-oka, Suita, Osaka 565-0871, Japan
E-Mail: tsuki(%)

Structural studies on membrane proteins have been performed at atomic level by both three-dimensional X-ray crystallography and two-dimensional electron crystallography in Japan as in Europe and Unites States. More than 13 membrane protein structures were elucidate by X-ray method in our country, and seven membrane protein structures were determined by cryo-electron microscopic method developed by Fujiyoshi of Kyoto University. Extensive crystallographic studies on calcium pump and cytochrome c oxidase elucidated their functional mechanisms at atomic level.

J. Cryst. Soc. Jpn., 52(1), 8-13 (2010).
Recent Progress and Development of Crystal Structure Analysis of Enzymes and Other Proteins
Masaru TANOKURA1 , Koji NAGATA2 , Ken-ichi MIYAZONO3 , Takuya MIYAKAWA4 and Masahiko OKAI5
Affilication: 1, 2, 3, 4, 5Graduate School of Agricultural and Life Sciences, University of Tokyo
Address: 1, 2, 3, 4, 51-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
E-Mail: 1amtanok(%), 2aknagata(%), 3amiyaz(%), 4atmiya(%), 5amokai(%)

Structural biology has made tremendous progress in this decade. Here we briefly introduce the Target Proteins Research Program, a national project promoted by the Ministry of Education, Culture, Sports, Science and Technology(MEXT)of Japan. The program aims to reveal the structure and function of proteins that are of great importance in both academic research and industrial application. We also summarize the results of structure-function analyses of(i)transcriptional regulatory proteins useful for the breading of drought and heat stress tolerant crops,(ii)useful enzymes for the production of chiral compounds, and(iii)useful enzymes for the degradation of environmental pollution substances. These results can be utilized in various areas of industries, to enhance food production, to improve the efficiency of pharmaceutical compound production, and to promote the bioremediation of contaminated soil and water.

J. Cryst. Soc. Jpn., 52(1), 14-18 (2010).
Ultra-high Resolution Protein Crystallography
Kazuki TAKEDA1 , Yu HIRANO2 and Kunio MIKI3
Affilication: 1, 2, 3Graduate School of Science, Kyoto University
Address: 1, 2, 3Sakyo-ku, Kyoto 606-8502, Japan
E-Mail: 1ktakeda(%), 2yuu(%), 3

Many protein structures have been determined by X-ray crystallography and deposited with the Protein Data Bank. However, these structures at usual resolution(1.5

J. Cryst. Soc. Jpn., 52(1), 20-24 (2010).
Structural Basis for Specific Recognition and Cleavage of Polyubiquitin Chains
Shuya FUKAI1 and Yusuke SATO2
Affilication: 1, 2Life Science Division, SRRO and IMCB, The University of Tokyo
Address: 1, 2218 General Research Bldg, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
E-Mail: 1fukai(%), 2yusato(%)

Ubiquitin is a conserved 76-residue protein that is reversibly conjugated with substrate proteins to regulate various cellular processes. The terminal carboxyl group of ubiquitin can be bonded to lysine residues of substrate proteins including ubiquitin itself. All seven lysine residues(K6, K11, K27, K29, K33, K48 and K63)and the N-terminal Met can act as receptors for the conjugation, producing polyubiquitin chains. For instance, the most abundant K48-linked chains serve as signals for proteasomal degradation, whereas K63-linked chains function in other proteasome-independent processes. Here we show how these functionally and structurally distinct chains are discriminated by linkage-specific deubiquitinating enzymes and binding proteins.

J. Cryst. Soc. Jpn., 52(1), 25-30 (2010).
Structure of the Gap Junction Channel
Affiliation: Institute for Protein Research, Osaka University
Address: 3-2 Yamadaoka, Suita, Osaka-fu 565-0871, Japan
E-Mail: maesyou(%)

Here we describe the long awaited atomic structure of the gap junction channel. The structure reveals intra-/inter- monomer interactions, which stabilize the channel structure, and intercellular interactions between two apposing hemichannels. The structure also reveals the pore structure in detail, charge distribution, pore-lining residues and so on. The novel structure, pore funnel, is found on the top of the pore and the relationship with channel gating could be inspected.

J. Cryst. Soc. Jpn., 52(1), 31-36 (2010).
Gibberellin Receptor GID1: Gibberellin Recognition and Molecular Evolution
Hiroaki KATO1 , Tomomi SATO2 and Miyako UEGUCHI-TANAKA3
Affilication: 1, 2Graduate School of Pharmaceutical Sciences, Kyoto University, 3Nagoya University, Bioscience and Biotechnology Center
Address: 1, 2Yoshida-Shimoadachi, Sakyo, Kyoto 606-8501, Japan, 3Chikusa-ku, Nagoya 464-8601, Japan
E-Mail: 1-mail:katohiro(%), 2tomomi(%), 3mueguchi(%)

Gibberellins(GAs)are phytohormones essential for many developmental processes in plants. We analyzed the crystal structure of a nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF 1(GID1)from Oryza sativa. As it was proposed from the sequence similarity, the overall structure of GID1 shows an a/b-hydrolase fold similar to that of the hormone-sensitive lipases(HSLs)except for an amino-terminal lid. The GA-binding site corresponds to the substrate-binding site of HSLs. Almost residues assigned for GA binding showed very little or no activity when they were replaced with Ala. The substitution of the residues corresponding to those of the lycophyte GID1s caused an increase in the binding affinity for GA34, a 2b-hydroxylated GA4. These findings indicate that GID1 originated from HSL and was tinkered to have the specificity for bioactive GAs in the course of plant evolution.

J. Cryst. Soc. Jpn., 52(1), 37-41 (2010).
Gibberellin Perception by the Gibberellin Receptor and its Effector Recognition
Toshio HAKOSHIMA1 , Kohji MURASE2 , Yoshinori HIRANO3 and Tai-ping SUN4
Affilication: 1, 2, 3Graduate School of Information Science, Nara Institute of Science and Technology, 4Department of Biology, Duke University
Address: 1, 2, 38916-5 Takayama, Ikoma, Nara 630-0192, Japan, 4専門分野:plant biology, gibberellin pathway
E-Mail: 1hakosima(%), 2, 3, 4

Gibberellins control a diverse range of growth and developmental processes in higher plants and have been widely utilized in the agricultural industry. By binding to a nuclear receptor GIBBERELLIN INSENSITIVE DWARF1(GID1), gibberellins regulate gene expression by promoting degradation of the transcriptional regulator DELLA proteins. The precise manner in which GID1 discriminates and becomes activated by bioactive gibberellins for specific binding to DELLA proteins remains unclear. We present the crystal structure of a ternary complex of Arabidopsis thaliana GID1A, a bioactive gibberellin and the N-terminal DELLA domain of GAI. In this complex, GID1a occludes gibberellin in a deep binding pocket covered by its N-terminal helical switch region, which in turn interacts with the DELLA domain containing DELLA, VHYNP and LExLE motifs. Our results establish a structural model of a plant hormone receptor which is distinct from the hormone-perception mechanism and effector recognition of the known auxin receptors.

J. Cryst. Soc. Jpn., 52(1), 43-47 (2010).
The Protein Micro-Crystallography Beamlines for Targeted Protein Research Program
Masaki YAMAMOTO1 , Naohiro MATSUGAKI2 and Soichi WAKATSUKI3
Affilication: 1RIKEN SPring-8 Center, 2, 3High Energy Accelerator Research Organization
Address: 11-1-1 Kouto, Sayo, Hyogo 679-5148, Japan, 2, 31-1 Oho, Tsukuba, Ibaraki 305-0801, Japan
E-Mail: 1yamamoto(%), 2naohiro.matsugaki(%), 3soichi.wakatsuki(%)

In order to collect proper diffraction data from outstanding micro-crystals, a brand-new data collection system should be designed to provide high signal-to noise ratio in diffraction images. SPring-8 and KEK-PF are currently developing two micro-beam beamlines for Targeted Proteins Research Program by MEXT of Japan. The program aims to reveal the structure and function of proteins that are difficult to solve but have great importance in both academic research and industrial application. At SPring-8, a new 1-micron beam beamline for protein micro-crystallography, RIKEN Targeted Proteins Beamline(BL32XU), is developed. At KEK-PF a new low energy micro-beam beamline, BL-1A, is dedicated for SAD micro-crystallography. The two beamlines will start operation in the end of 2010. The present status of the research and development for protein micro-crystallography will be presented.

J. Cryst. Soc. Jpn., 52(1), 48-51 (2010).
Neutron Crystallography for Macromolecular Structure Analysis
Affiliation: Quantum Beam Science Directorate, Japan Atomic Energy Agency
Address: 2-4 Shirakata-shirane, Tokai, Ibaraki 319-1195, Japan
E-Mail: kuroki.ryota(%)

Hydrogen atoms in proteins as well as protein-bound water molecules play a significant role in many chemical reaction processes in living systems, such as catalytic reaction and molecular recognition. Neutron crystallography is a powerful tool to identify locations of light atoms like hydrogen. In the field of neutron crystallography, the development of diffractometers and techniques for preparation and crystallization of target samples has been developed to complement the low flux of neutron sources. In Japan, single-crystal diffractometers named BIX-3 and BIX-4 have been developed, and contribute to the effective collection of neutron diffraction data. Recent developments on the complementary use of neutron and X-ray diffraction data have begun solving previously undetermined problems of protein function. Further efforts to acquire higher measurement performance are now in progress to increase the application of neutron crystallographic studies.

J. Cryst. Soc. Jpn., 52(1), 52-55 (2010).
Coarse-Grained Structural Dynamics of Proteins Revealed by Solution Scattering
Affiliation: Department of Biomolecular Science, Faculty of Engineering, Gifu University
Address: 1-1 Yanagido, Gifu 501-1193, Japan
E-Mail: fujisawa(%)

Small-angle scattering(SAS)from protein solutions gives low-resolution structural information about protein folding, extended conformations, flexibility linked domains, allosteric change of oligomer proteins, and assembly/disassembly of protein complexes. This review deals with the introduction of recent progress of SAS modeling and the future prospect of SAS technique as well.

J. Cryst. Soc. Jpn., 52(1), 56-61 (2010).
Cryo-Electron Microscopy of Biological Macromolecular Structures
Affiliation: RIKEN SPring-8 Center
Address: 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan
E-Mail: yone(%)

There are many huge macromolecular complexes in living organisms. They are often hard to crystallize because of their size, complexity and heterogeneity. Cryo-electron microscopy(cryo-EM)is a suitable method to analyze the structures of such biological macromolecules, because it can be applied to various forms of samples, e.g. two-dimensional crystal, helical assembly, spherical virus, dispersed particle, cell organelle and cell, although attainable resolution depends on the system. In this review, I introduce these techniques and examples of the structure analysis, and briefly review the perspective of cryo-EM.

J. Cryst. Soc. Jpn., 52(1), 62-67 (2010).
Application of the Maximum Entropy Method in the Macoromolecular Crystallography
Affiliation: 〒464-8603 名古屋市千種区不老町1
Address: e-mail:
E-Mail: eiji(%)

Accurate structural refinement of a putative acylphoshatase from 1.3 ・X-ray diffraction data was carried out using charge densities determined by the Maximum Entropy Method(MEM). The MEM charge density clearly revealed detailed features in the solvent region of the putative acylphosphatase crystalline structure, some of which have never been seen in conventional Fourier map. The structural model in solvent region was constructed as distributions of anisotropic water atoms. The omit-MEM maps and the difference-MEM maps were effective for revealing details of protein structure, such as multi-conformers in side-chains of amino acid residues, anisotropy of atoms, and hydrogen atoms. By model building using the MEM charge densities, the reliability factors, R1 and Rfree, in the SHELX refinements were improved to 9.6% and 10.0%, respectively.

J. Cryst. Soc. Jpn., 52(1), 69-75 (2010).
Recent Advances in Biology of Cysteinyl Leukotriene
Hideo AGO1 and Masashi MIYANO2
Affilication: 1, 2RIKEN SPring-8 Center, Structural Biophysics Laboratory
Address: 1, 21-1-1 Kouto, Sayo-Cho, Sayo-Gun, Hyogo 678-5148, Japan
E-Mail: 1ago(%), 2miyano(%)

Cysteinyl leukotriene has been known to be a major component of SRS-A(Slow Reacting Substance of Anaphylaxis). Inhibitors affecting on its biosynthesis and antagonists of its G-protein coupled receptor are therapeutic agents for acute inflammatory diseases, such as bronchial asthma and rhinitis. Latest findings in pathobiology of asthma suggest that cysteinyl leukotriene would take a key role in not only acute but also chronic inflammation of asthma. The structure basis of biosynthesis of cysteinyl leukotriene was revealed by crystallographic analysis of human membrane protein leukotriene C4 synthase.

J. Cryst. Soc. Jpn., 52(1), 76-80 (2010).
Membrane-Protein Crystallography and Potentiality for Drug Design −An Example from Neurotransmitter Transporter Homolog LeuT
Affiliation: RIKEN SPring-8 Center
Address: 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan
E-Mail: atsuko(%)

Structure-based drug design for membrane proteins is far behind that for soluble proteins due to difficulty in crystallographic structure determination, despite the fact that about 60% of FDA-approved drugs target membrane proteins located at the cell surface. Stable homologs for a membrane protein of interest, such as prokaryotic neurotransmitter transporter homolog LeuT, might enable cooperative analyses by crystallography and functional assays, provide useful information for functional mechanisms, and thus serve as important probes for drug design based on mechanisms as well as structures.

J. Cryst. Soc. Jpn., 52(1), 81-88 (2010).
Creation of High Efficient Firefly Luciferase
Affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University
Address: 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
E-Mail: nakatsu(%)

Firefly emits visible yellow-green light. The bioluminescence reaction is carried out by the enzyme luciferase. The bioluminescence of luciferase is widely used as an excellent tool for monitoring gene expression, the measurement of the amount of ATP and in vivo imaging. Recently a study of the cancer metastasis is carried out by in vivo luminescence imaging system, because luminescence imaging is less toxic and more useful for long-term assay than fluorescence imaging by GFP. However the luminescence is much dimmer than fluorescence. Then bioluminescence imaging in living organisms demands the high efficient luciferase which emits near infrared lights or enhances the emission intensity. Here I introduce an idea for creating the high efficient luciferase based on the crystal structure.

J. Cryst. Soc. Jpn., 52(1), 89-94 (2010).
The Trial of Drug Discovery using the In-Silico Screening Methods Developed by Pharmaceutical Innovation Value Chain
Tsuyoshi INOUE1 , Hiroyoshi MATSUMURA2 , Hiroaki ADACHI3 , Yusuke MORI4 , Kazufumi TAKANO5 , Yoshifumi FUKUNISHI6 , Haruki NAKAMURA7 , Takayoshi KINOSHITA8 , Isao NAKANISHI9 , Satoshi MINAKATA10 , Yoshiaki MIKAMI11 , Toshihiro SAKUMA12 , Masato KITAJIMA13 , Yoshitada FUKUOKA14 , Toshikazu TAKADA15 and Tsuneaki SAKATA16
Affilication: 1, 2, 4, 5, 10Graduate School of Engineering, Osaka University, 3SOSHO, Inc., 6Protein Structural Information Analysis Team Biomedicinal Information Research Center(BIRC)National Institute of Advanced Industrial Science and Technology(AIST), 7Institute for Protein Research, Osaka University, 8Graduate School of Science, Osaka Prefecture University, 9Faculty of Pharmacy, Kinki University, 11Hitachi East Japan Solutions, Ltd., 12NEC Informatec Systems, Ltd., 13Fujitsu Kyushu Systems, Ltd., 14Mitsui Knowledge Industry, Ltd., 15RIKEN Computational Science Research Program, 16Cybermedia Center, Osaka University
Address: 1, 2, 4, 5, 102-1 Yamadaoka, Suita, Osaka 565-0871, Japan, 3大阪大学先端科学イノベーションセンターA207号, 6Aomi 2-3-26, Koto-ku, Tokyo 135-0064, Japan, 73-2 Yamadaoka, Suita, Osaka 565-0871, Japan, 81-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan, 93-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan, 1112-1 Ekimae-honcho, Kawasaki-ku, Kawasaki, Kanagawa 210-0007, Japan, 122-6-1 Kitamikata, Takatsu-ku, Kawasaki, Kanagawa 213-8511, Japan, 132-2-1 Momochihama, Sakura-ku, Fukuoka 814-8589, Japan, 142-3-33 Nakanoshima, Kita-ku, Osaka 530-0005, Japan, 152-1 Hirosawa, Wako, Saitama 351-0198, Japan, 16Urban Innovation Institute, 1-12-39 Umeda, Kita-ku, Osaka 530-0001, Japan
E-Mail: 1inouet(%), 2matsumura(%), 3adachi(%), 4mori.yusuke(%), 5ktakano(%), 6y-fukunishi(%), 7harukin(%), 8kinotk(%), 9isayan(%), 10minakata(%), 11, 12t-sakuma(%), 13kitajima.masato(%), 14fukuoka-yoshitada(%), 15tz-takada(%), 16tsuneaki.sakata(%)

We have recently established Pharmaceutical Innovation Value Chain collaborated by The SOSHO project( The BioGrid Project( accelerate new drug development. The in-silico group calculated the matrices on the interaction between the proteins and chemical compounds, and developed the novel in-silico screening methods, Multiple Target Screening(MTS)and Docking score index(DSI), improving the hit rate of screening a lead compound.

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