• 1974 Bachelor of Engineering Science (Biophysics) Department of Biophysical Engineering, Osaka University, Toyonaka, Osaka
• 1976 Master of Engineering Science (Biophysics) Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka
• 1980 Ph.D. (Biophysics) Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka

• 1980-1981 Postdoctral Fellow (Japan Society for the Promotion of Science) Dept. of Biophysical Engineering, Osaka University, Toyonaka, Osaka
• 1981-1984 Research Associate, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA
• 1984-1985 Research Associate, Department of Molecular Biology, Vanderbilt University, Nashville, TN
• 1985-1986 Research Instructor, Department of Molecular Biology, Vanderbilt University, Nashville, TN
• 1986 Senior Research Associate, Rosenstiel Basic Medical Sciences, Research Center, Brandeis University, Waltham, MA
• 1986-1991 Group Leader, Molecular Dynamic Assembly Project, ERATO, JRDC, Tsukuba, Ibaragi
• 1991 Research Manager, Molecular Dynamic Assembly Project, ERATO, JRDC, Tsukuba, Ibaragi and Kyoto, Kyoto
• 1992-1999 Research Director, International Institute for Advanced Research, Matsushita Electric Industrial Co., Ltd., Seika, Kyoto
• 1997-2003 Project Director, Protonic NanoMachine Project, ERATO, JST, Seika, Kyoto
• 1999-2002 Research Director, Advanced Technology Research Laboratories, Matsushita Electric Industrial Co., Ltd., Seika, Kyoto
• 2001-2002 Group Leader, Structural Biology Group, Japan Synchrotron Radiation Research Institute (SPring-8), Harima, Hyogo
• 2002- Professor, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka
• 2002- Visiting Chief Researcher, Japan Synchrotron Radiation Research Institute (SPring-8), Harima, Hyogo
• 2002- Project Director, Dynamic NanoMachine Project, ICORP, JST, Seika, Kyoto

• Biophysical Society of Japan; Molecular Biology Society of Japan; Japan
• Protein Science Society, Biophysical Society; Microscopy Society of America;
• American Association for the Advancement of Science

• 2001 Osaka Science Prize
• 2002 Visual Science Festa 2002 Distinguished Award
• 2003 TEPIA High-Tech Video Concours TEPIA Grand Prix

Background: Namba Protonic NanoMachine Project studied the structure and function of the bacterial flagellum as a huge molecular system based on the organization and movement of the individual atoms that build it.

Research Results:

Crystallization of flagellar proteins: To determine the molecular structures of flagellin to identify the mechanism of the mechanical switch of high precision and of many other flagellar proteins to understand how they work, they were cloned, overproduced, purified and crystallized. For fiber-forming proteins, it was first necessary to produce fragments that do not polymerize by removing the terminal regions that stabilize the fiber structure. X-ray crystallography: Atomic models of fragments of flagellin, hook protein, and HAP3 were built at around 2 A resolution from diffraction data obtained by using brilliant X-rays from SPring-8 beam-lines. Crystals of other flagellar proteins yielded high-resolution diffraction data as well. The a-axis molecular array in the flagellin crystal was identified to be the flagellar protofilament with the shorter repeat distance of its two distinct conformations, whose repeat distance is 51.9 A and 52.7 A. Docking of the molecular array into a density map of the flagellar filament obtained by electron cryomicroscopy also proved it.

Mechanical switch: A computer simulation of the protofilament extension was carried out to find the flagellin structure responsible for the high-precision mechanical switch. This work greatly stimulated the research of the mechanical properties of proteins.

Electron cryomicroscopy and image analysis: Electron cryomicroscopy and image analysis were used to analyze the flagellar filament or the cap-filament complex. Thousands of images were collected and carefully processed, one by one, and either helical image reconstruction or single-particle image analysis was used to reconstruct the three-dimensional images at high-resolution.

Flagellar cap as assembly nanomachine: Flagellin subunits are arranged in a helical manner to form the filament with approximately 5.5-fold symmetry, but the cap bound at its distal end is a HAP2 pentamer with 5-fold symmetry, having a pentagonal table top with five leg domains. The molecular structure revealed a rotary mechanism of the cap to promote self-assembly of flagellin to the filament-end, which is achieved by its binding over the symmetry-mismatch.

Structure analysis of motor: Structural studies of the motor protein complexes were also conducted by electron cryomicroscopy and X-ray crystallography to understand how the motor produces the torque by proton flow even at an energy level comparable to the thermal noise, and why the bushing works well without lubrication. Motor proteins having membrane-spanning structures were overproduced and new purification methods were developed.

Flagellar motor rotation: To understand the mechanism of the highly efficient proton motor, the rotation speed and its fluctuation or stepping motion were examined by nanophotometry. A new optical system incorporating a specially designed high-sensitivity quadrant photo-sensor was developed for measuring single flagellar motor rotation at high temporal and spatial resolution. Data indicate that the motor rotates with unexpectedly large, rapid fluctuations and even stops occasionally for several milliseconds.

1. Maki-Yonekura, S., Yonekura, K. and Namba, K. (2003)
   Domain movements of HAP2 in the cap-filament complex formation and growth process of the bacterial flagellum.
   Proc. Natl. Acad. Sci. USA 100, 15528-15533.
2. Yonekura, K., Maki-Yonekura, S. and Namba, K. (2003)
   Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy.
   Nature 424, 643-650.
3. Hayashi, F., Suzuki, H., Iwase, R., Uzumaki, T., Miyake, A., Shen, J.R., Imada, K., Furukawa, Y., Yonekura, K., Namba, K. and Ishiura, M. (2003)
   ATP-induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC.
   Genes Cells 8, 287-296.
4. Samatey, F.A., Imada, K., Nagashima, S., Vonderviszt, F., Kumasaka, T., Yamamoto, M. and Namba, K. (2001)
   Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling.
   Nature 410, 331-337.
5. Namba, K. (2001)
   Roles of partly unfolded conformations in macromolecular self-assembly.
   Genes Cells 6, 1-12.
6. Yonekura, K., Maki, S., Morgan, D.G., DeRosier, D.J., Vonderviszt, F., Imada, K. and Namba, K. (2000)
   The bacterial flagellar cap as the rotary promoter of flagellin self-assembly.
   Science 290, 2148-2152.

Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism

FADEL A. SAMATEY^1,2,3, HIDEYUKI MATSUNAMI^1,2,3, KATSUMI IMADA^1,2,3, SHIGEHIRO NAGASHIMA^1,3, TANVIR R. SHAIKH^4,*, DENNIS R. THOMAS⁴, JAMES Z. CHEN⁴, DAVID J. DEROSIER⁴, AKIO KITAO⁵ & KEIICHI NAMBA^1,2,3

¹ Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
² Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
³ Protonic NanoMachine Project, ERATO, JST, 3-4 Hikaridai, Seika, Kyoto 619-0237, Japan
⁴ W. M. Keck Institute of Cellular Visualization, Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110, USA
⁵ Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-0032, Japan
^* Present address: Wadsworth Center, P.O. Box 509, Albany, New York 12201, USA

Correspondence and requests for materials should be addressed to K.N. (keiichi@fbs.osaka-u.ac.jp).
Atomic coordinates have been deposited in the Protein Data Bank under accession code 1WLG.

The bacterial flagellum is a motile organelle, and the flagellar hook is a short, highly curved tubular structure that connects the flagellar motor to the long filament acting as a helical propeller. The hook is made of about 120 copies of a single protein, FlgE, and its function as a nano-sized universal joint is essential for dynamic and efficient bacterial motility and taxis. It transmits the motor torque to the helical propeller over a wide range of its orientation for swimming and tumbling. Here we report a partial atomic model of the hook obtained by X-ray crystallography of FlgE31, a major proteolytic fragment of FlgE lacking unfolded terminal regions, and by electron cryomicroscopy and three-dimensional helical image reconstruction of the hook. The model reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function.