FBS Colloquia No.413JEOL YOKOGUSHI Research Alliance Laboratories
| Seminar or Lecture |
Cryo-EM structure of the FlhA ring in a functional export state Miki Kinoshita [Specially Appointed Assistant Professor, JEOL YOKOGUSHI Research Alliance Laboratories] Direct imaging of microcrystal structures by Precession Illumination in a transmission electron cryomicroscope Keiichi Namba [Specially Appointed Professor, JEOL YOKOGUSHI Research Alliance Laboratories] |
|---|---|
| Date and Time | 19 May 2026 (Tue), 12:15～13:00 |
| Place | 2F Seminar Room, BioSystems Building |
| Language | Japanese |
| Contact |
Tomoko Miyata (Specially Appointed Associate Professor) |
Cryo-EM structure of the FlhA ring in a functional export state
In this colloquium, I will provide an overview of structural studies on the bacterial flagellar motor in Salmonella. The flagellum is a macromolecular machine responsible for bacterial motility, and its assembly requires a dedicated protein export apparatus located at the base. Structural studies have revealed the architectures of multiple components of the flagellum, including the MS ring, the filament, and the FliPQR complex that forms the core of the export apparatus. I will first summarize recent advances in these structural studies and then focus on our recent cryo-electron microscopy (cryo-EM) analysis of the FlhA ring. FlhA is a central component that regulates substrate selection and the export order of flagellar proteins. Its cytoplasmic domain forms a nonameric ring that serves as a docking platform for export substrates, whereas its transmembrane domain functions as an ion channel that drives protein export. Using single-particle cryo-EM analysis, we determined a high-resolution structure of the FlhA ring in a hook-type export state by analyzing a linker deletion mutant that connects the transmembrane and cytoplasmic domains. This structure reveals, for the first time, the overall architecture of the FlhA ring including the transmembrane region. Notably, the FHIPEP region is positioned on the outer side of the ring, in a configuration that markedly differs from predictions by AlphaFold. These findings provide structural insights into how conformational changes in FlhA contribute to the stage-specific regulation of flagellar protein export.
Direct imaging of microcrystal structures by Precession Illumination in a transmission electron cryomicroscope
We will present our latest results from a collaborative project with Prof. Tsutomu Shintake at the Okinawa Institute of Science and Technology. Protein crystals are three-dimensional arrangements of molecules with translational symmetry. When observed from specific angles, the molecules overlap to produce high-contrast images; however, since image formation results from the interference of diffracted waves and incident waves, the diffraction conditions in reciprocal space (Fourier space) are satisfied only near the center of the reciprocal plane due to the curvature of the Ewald sphere. Consequently, the resolution of the projected image is limited. In this study, through specialized modifications to a transmission electron cryomicroscope, we achieved direct observation of crystal structure images by enabling precession illumination via the tilt of the incident electron beam and selective extraction of diffracted waves on specific reciprocal lattice plane through the introduction of a ring slit. Using this method, we successfully observed high-resolution real images, such as molecular arrangements within protein crystals and atomic arrangements of organic molecules in perovskites. This technique is also applicable to microscopic mosaic crystals and is expected to find applications in drug discovery and the development of functional materials.
