The EM maps of the four conformations were deposited to Electron Microscopy Data Bank (EMDB) as accession codes EMD-9845 (open), EMD-9846 (half-open1), EMD-9847 (half-open2), and EMD-9848 (closed), respectively. In addition, the structural models built for the Abbreviations AFM, atomic force microscopy; core domain, core domain engaging in hexamer formation; cryoEM, cryo-electron microscopy; FSC, Fourier shell correlation; GDH, glutamate dehydrogenase; GS-FSC, gold-standard Fourier shell correlation; MD, molecular dynamics; NAD domain, nucleotide-binding domain; PC, principal component; PCA, principal component analysis; R g , radius of gyration; SAXS, small-angle X-ray scattering; T. profundus, Thermococcus profundus; tris, hydroxymethyl aminomethane.

Coherent X-ray diffraction imaging (CXDI) allows internal structures of biological cells and cellular organelles to be analyzed. CXDI experiments have been conducted at 66 K for frozen-hydrated biological specimens at the SPring-8 Angstrom Compact Free-Electron Laser facility (SACLA). In these cryogenic CXDI experiments using X-ray free-electron laser (XFEL) pulses, specimen particles dispersed on thin membranes of specimen disks are transferred into the vacuum chamber of a diffraction apparatus. Because focused single XFEL pulses destroy specimen particles at the atomic level, diffraction patterns are collected through raster scanning the specimen disks to provide fresh specimen particles in the irradiation area. The efficiency of diffraction data collection in cryogenic experiments depends on the quality of the prepared specimens. Here, detailed procedures for preparing frozenhydrated biological specimens, particularly thin membranes and devices developed in our laboratory, are reported. In addition, the quality of the frozen-hydrated specimens are evaluated by analyzing the characteristics of the collected diffraction patterns. Based on the experimental results, the internal structures of the frozen-hydrated specimens and the future development for efficient diffraction data collection are discussed.

X-ray diffraction imaging is a technique for visualizing the structure of biological cells. In X-ray diffraction imaging experiments using synchrotron radiation, cryogenic conditions are necessary in order to reduce radiation damage in the biological cells. Frozen-hydrated biological specimens kept at cryogenic temperatures are also free from drying and bubbling, which occurs in wet specimens under vacuum conditions. In a previous study, the diffraction apparatus KOTOBUKI-1 , Rev. Sci. Instrum. 84, 093705] was constructed for X-ray diffraction imaging at cryogenic temperatures by utilizing a cryogenic pot, which is a cooling device developed in low-temperature physics. In this study a new cryogenic pot, suitable for tomography experiments, has been developed. The pot can rotate a biological cell over an angular range of AE 170 against the direction of the incident X-ray beam. Herein, the details and the performance of the pot and miscellaneous devices are reported, along with established experimental procedures including specimen preparation. The apparatus has been used in tomography experiments for visualizing the threedimensional structure of a Cyanidioschyzon merolae cell with an approximate size of 5 mm at a resolution of 136 nm. Based on the experimental results, the necessary improvements for future experiments and the resolution limit achievable under experimental conditions within a maximum tolerable dose are discussed.research papers

Phototropin1 is a blue light (BL) receptor in plants and shows BL-dependent kinase activation. The BL-excited light-oxygenvoltage-sensing domain 2 (LOV2) is primarily responsible for the activation of the kinase domain; however, the molecular mechanism by which conformational changes in LOV2 are transmitted to the kinase domain remains unclear. Here, we investigated BL-induced structural changes of a minimum functional fragment of Arabidopsis phototropin1 composed of LOV2, the kinase domain, and a linker connecting the two domains using small-angle x-ray scattering (SAXS). The fragment existed as a dimer and displayed photoreversible SAXS changes reflected in the radii of gyration of 42.9 Å in the dark and 48.8 Å under BL irradiation. In the dark, the molecular shape reconstructed from the SAXS profiles appeared as two bean-shaped lobes in a twisted arrangement that was 170 Å long, 80 Å wide, and 50 Å thick. The molecular shape under BL became slightly elongated from that in the dark. By fitting the crystal structure of the LOV2 dimer and a homology model of the kinase domain to their inferred shapes, the BL-dependent change could be interpreted as the positional shift in the kinase domain relative to that of the LOV2 dimer. In addition, we found that lysine 475, a functionally important residue, in the N-terminal region of LOV2 plays a critical role in transmitting the structural changes in LOV2 to the kinase domain. The interface between the domains is critical for signaling, suitably changing the structure to activate the kinase in response to conformational changes in the adjoining LOV2. Phototropin (phot)2 is one of the blue light (BL) receptor proteins in plants (1, 2). It plays several important roles to maximize the efficiency of photosynthesis: namely phototropism (3), chloroplast movement (4, 5), stomata opening (6), and leaf expansion (7). Most plants possess two isoforms of phot, designated as phot1 and phot2 (1). Both isoforms redundantly regulate stomata opening (6) and chloroplast accumulation depending on fluence rate of BL (3). However, only phot2 is responsible for the photoavoidance response in chloroplast relocation (4). Regarding the phototropic response, phot2 works under high irradiance condition, whereas phot1 predominantly initiates the response under low irradiance condition (3).The structure of plant phot, which consists of ϳ950 -1000 amino acid residues and two flavin mononucleotide (FMN) molecules (1), folds into three functional domains (Fig. 1A). The N-terminal half contains two light-oxygen-voltage-sensing domains (LOVs) (designated LOV1 and LOV2) (8, 9), which belong to the PAS (Per-Arnt-Sim) superfamily found in heterodimeric interaction in cellular signaling. Each LOV domain receives BL via the FMN molecule bound to the pocket formed by the characteristic ␣-helix/␤-strand-mixed structure known as the ␣/␤-scaffold (10, 11). The C-terminal half of phot is a serine-threonine kinase (STK) domain classified into group VIII of the AGC family (12). The linker region, which has J␣-heli...