Journal of the Crystallographic Society of Japan
Volume 45 (1), 2003.

J. Cryst. Soc. Jpn., 45(1), 3-8, (2003).
Cell-free Protein Synthesis by Wheat Germ Extracts
Yuzuru TOZAWA1, Tatsuya SAWASAKI2 and Yaeta ENDO3
Affiliation: 1Mitsubishi Kagaku Institute of Life Sciences, 2,3Faculty of Engineering, Ehime University
Address: 11000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-8502, Japan, 2,33 Bunkyo-cho, Matsuyama 790-8577, Japan

With the sequencing of the genomes of various species, attention has turned to the structure, properties, and functional activities of proteins. However, rapid progress in the area of proteomics is premised on the availability of sufficient amounts of a large number of proteins. Here we described a novel cell-free system from wheat embryos for the high-throughput screening/synthesis of gene products. Our system should open up many possibilities in the post-genome era.

J. Cryst. Soc. Jpn., 45(1), 9-13, (2003).
Preparation of High Resolution Crystals Based on Crystallogenesis |The Case of Cytochrome c|
Mitsuo ATAKA
Affiliation: National Institute of Advanced Industrial Science and TechnologyiAISTj
Address: AIST, Kansai, Ikeda 563-8577, Japan

A basis for protein crystal preparation for high resolution structure determination is provided for vertebrate cytochrome c. Crystals are shown to grow in the presence of ammonium sulfate and sodium nitrate for tuna, horse and bovine cytochrome c. All the crystals obtained diffracted X-ray to a resolution better than 2.0ð. X-ray crystallography revealed that a nitrate ion combines two protein molecules near the C-terminal, thus stabilizing the terminal region. A combined use of two salts for crystallization is considered to be a reasonable and rationalized way of growing high quality crystals for this protein.

J. Cryst. Soc. Jpn., 45(1), 14-18, (2003).
Development of Structure Analysis by MAD Method
Takashi KUMASAKA1 and Masaki YAMAMOTO2
Affiliation: 1Department of Life Science, Tokyo Institute of Technology, 2X-ray Coherent Optics Laboratory, RIKEN Harima Institute
Address: 14259 Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan, 21-1-1 Kouto, Mikazuki, Sayo, Hyogo 678-1205, Japan

The crystallographic phase problem had not been routinely solved in protein crystallography. Nowadays, MAD method is widely used for crystallographic determination of unknown protein structures, since the scope of MAD application has been extended by the use of synchrotron radiation facilities, protein expression techniques, and computational development. The method has paved the way to high-throughput structure determination of protein crystals and introduced to structural genomics study. We describe here a brief summary of MAD method and its experimental.

J. Cryst. Soc. Jpn., 45(1), 19-25, (2003).
Molecular Mechanisms of Multi-protein Complex Formations on Promoter DNA and Transcriptional Regulations
Kazuhiro OGATA
Affiliation: Department of Biochemistry, Yokohama City University School of Medicine
Address: 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan

In the eukaryotic cells, the gene expressions are regulated by multiple transcriptional regulatory factors bound to the promoter regions of the target genes. The DNA-binding activity and trans-activating capacity of these transcription factors are precisely controlled via synergistic intermolecular interactions. Based on the structural analyses using X-ray crystallography, atomic force microscopy and temperature-scanning spectroscopy and the functional analyses using various molecular interaction experimentsiincluding GST-pull down assay, gel shift assay, calorimetry and surface plasmon resonance experimentsjand trans-activation assays, the underlying molecular mechanisms of the transcriptional regulations in the hematopoietic system and their deregulated states in the leukemic cells are presented.

J. Cryst. Soc. Jpn., 45(1), 26-31, (2003).
Structural Biology of Protein Transport
Affiliation: Photon Factory, Institute of Materials Structure Science, KEK
Address: 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan

Many eukaryiotic proteins are glycosylated after translation to become mature proteins. Efficient sorting of such glycosylated proteins is essential for the cell's function and is achieved by vesicle transport. Mannose 6-phosphateiM6Pjmodification of lysosomal proteins is recognized by a M6P receptoriMPRjwhich incorporates the lysosomal proteins to vesicles. Recently GGA proteins have been identified and later verified as a new family of adapter proteins distinct from well known AP-1 to 4 complexes. They are responsible for vesicle transport of glycosylated human proteins from the Golgi apparatus to early endosomes/lysosomes. We have determined the crystal structures of the VHS domain of human GGA1 and the ear domain of human g1 adaptin. Combined with biochemical and cell-biological data, these structures reveal the recognition mechanisms of the acidic dileucine motif signal of mannose-6-phophate receptor by the VHS domain of human GGA protein and the interaction between the ear domain of g1 adaptin of the AP-1 complex and g-synergin and Rabaptin-5. They constitute part of a rapidly growing ensemble of structures of transport proteins, where protein-protein interactions, as elucidated by X-ray structural analyses, play critical roles.

J. Cryst. Soc. Jpn., 45(1), 32-36, (2003).
From Complex Structure to Dynamic Properties
Yuichiro MAEDA
Affiliation: RIKEN Harima Institute at SPring-8
Address: 1-1-1 kouto, Mikaduki, Sayo, Hyogo 679-5148

The actin filament is a flexible and easy-to-idis-jassemble structure. In order to understand the mechanism of calcium regulation of muscle contraction, we have recently obtained the crystal structure of troponin, which implied that troponin may regulate the flexibility of actin /tropomyosin filament. The questions to address are, on one hand, how to preparegmini-actin filamentshsuitable for crystallization, and on the other, how to deduce dynamic properties of the actin filament from the static structure.

J. Cryst. Soc. Jpn., 45(1), 37-42, (2003).
Structural Study of the Bacterial Flagellar Motor System as a Molecular Nano-machine
Katsumi IMADA1, Fadel A. SAMATEY2 and Keiichi NAMBA3
Affiliation: Graduate School of Frontier Biosciences, Osaka University
Address: 3-4 Hikaridai, Seika, Kyoto 619-0237, Japan
E-mail: , ,

The bacterial flagellum is a motility apparatus in which a helical filament is driven by a rotary motor. The long helical filament, which acts as a screw, is not a rigid propeller, but switches its helical form upon quick reversal of the motor rotaion. Complementary use of X-ray diffraction and electron cryomicroscopy reveals the detail structure of this large complex. We describe the molecular mechanism of polymorphic supercoiling based on the structure.

J. Cryst. Soc. Jpn., 45(1), 43-47, (2003).
The Ribosome, Translation Machinery
Min YAO1, Takashi NAKASHIMA2 and Isao TANAKA3
Affiliation: Division of Biological Sciences, Graduate School of Science, Hokkaido University
Address: Kita 8 Nishi 5, Kita-ku, Sapporo 060-0810, Japan

The ribosome is a ribonucleoprotein complex responsible for protein synthesis. The three-dimensional structures of the ribosome subunits have elucidated the mechanisms of this translation machinery in molecular basis. The relationships between the proteins and RNAs in this ribonucleoprotein complex are discussed.

J. Cryst. Soc. Jpn., 45(1), 48-53, (2003).
Structural Analysis of the Baseplate of Bacteriophage T4 and Elucidation of the Infection Mechanism
Shuji KANAMARU1, Petr LEIMAN2 and Fumio ARISAKA3
Affiliation: 1,2Department of Biological Sciences, Purdue University, 3Department of Molecular and Cellular Assembly Graduate School of Bioscience and Biotechnology Tokyo Institute of Technology
Address: 1,2915 W. State St., West Lafayette, IN 47907-2054, 34259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan

The sub-atomic structure of the tail-lysozyme complex of bacteriophage T4 has been determined to the resolution of 2.9ð. For the phase determination, MADimulti-wavelength anomalous dispersionjfrom seleno-methionine-substituted gp27 which had been complexed with unlabeled gp5igpgene productjwas utilized. The tail-lysozyme was then localized in the low-resolution structure of the tail baseplate, which revealed the role of the C-terminal ƒÀ-helix domain as a cell-puncturing device as well as an intra-molecular chaperone to form the trimeric tail-lysozyme complex.

J. Cryst. Soc. Jpn., 45(1), 54-59, (2003).
Biological Machinery in Higher-Order Structure Formation System of Proteins
Yasuhito SHOMURA1 and Kunio MIKI2
Affiliation: 1Department of Chemistry, Graduate School of Science, Kyoto University, 2Graduate School of Science, Kyoto University / RIKEN Harima InstituteiSPring-8j
Address: 1Sakyo-ku, Kyoto 606-8502, Japan, 2Sakyo-ku, Kyoto 606-8502, JapaniKyoto Univ.j

The folding of protein molecules and the maintenance of their biological functions are controlled by molecular chaperons, which are good targets for structural biology of biological machinery. Crystal structures and structural conversion of chaperonin systemigroups I and IIjare reviewed as a typical example of structural biology of molecular chaperones and a view for further crystallographic studies in this field is discussed.

J. Cryst. Soc. Jpn., 45(1), 60-65, (2003).
Crystal Structure Analysis and the Mechanism of Active Transport by the Calcium Pump of Sarcoplasmic Reticulum
Chikashi TOYOSHIMA1 and Hiromi NOMURA2
Affiliation: Institute of Molecular and Cellular Biosciences, The University of Tokyo
Address: 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

Calcium pumpiCa2+-ATPasejof muscle sarcoplasmic reticulum is an integral membrane protein of Mr 110 k and a representative member of P-type ATPases involved in the active transport of ions across the membrane using the energy liberated by the hydrolysis of ATP. Crystal structures of this enzyme has now been determined for both calcium bound and unbound states. The structures exhibit a very large scale domain movements between the two states and provide insight into the mechanism of active transport. These pumps appear to work in a similar way to mechanical pumps at an atomic scale.

J. Cryst. Soc. Jpn., 45(1), 66-70, (2003).
Research on Biological Machinery of Bioluminescence
Masaru TANOKURA1 and Woo-Cheol LEE2
Affiliation: Graduate School of Agricultural and Life Sciences, University of Tokyo
Address: 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

The living organism is a complex system consisting of numerous biological machineries that are defined as a functional unit responsible for particular biological activities, such as protein synthesis, ATP synthesis, and various kinds of metabolisms. Biological machineries may be classified into two types. The first one refers to stable assembly systems of macromolecules. This kind of machineries may sometimes be crystallized and their whole structures are determined by the X-ray crystallography. The second one does not create such a stable assembly system, but by the concerted acts of the individual macromolecules, the systems play as whole complex biological roles. The development of protein crystallography in recent years makes it possible to determine the overall three-dimensional structures of the biological machinery of both types. Three-dimensional structure determination of biological machineries would open a new era in the field of structural biology.

J. Cryst. Soc. Jpn., 45(1), 71-75, (2003).
Structural Biology of Dioxygenases in Polychlorinated-biphenyliPCBjDegradation Pathway
Toshiya SENDA1 and Masao FUKUDA2
Affiliation: 1Biological Information Research Center / National Institute of Advanced Industrial Science and Technology, 2Department of BioEngineering, Nagaoka University of Technology
Address: 12-41-6 Aomi, Koto-ku, Tokyo 135-0064, JAPAN, 2Kamitomioka, Ngaoka, Niigata 940-2188, JAPAN

Two dioxygenases found in the PCB degradation pathway, biphenyl dioxygenase and extradiol type catecholic dioxygenase, play key roles in the pathway. The crystal structures of reaction intermediates of the dioxygenases have revealed the catalytic mechanisms of theses enzymes.

J. Cryst. Soc. Jpn., 45(1), 76-80, (2003).
Crystal Structure Determination of Biological Macromolecular Assemblies; From Data Collection to Structure Determination
Affiliation: Research Center for Structural and Functional Proteomics, Institute for Protein Research, Osaka University
Address: 3-2 Yamadaoka, Suita, Osaka 565-8701, Japan

Recent progresses in crystallography on biological macromolecules enable us to solve the atomic structure of the huge macromolecule assemblies. High-brilliance synchrotron radiation and a high-performance area detector are essential for high-quality diffraction data collection from a small-size crystal. Newly developed software for crystal structure determination can be used for quick and accurate structure determinaion. In this paper, we will review the recent progress in crystallography on biological macromolecular assemblies

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