SBRC is a leading research center of structural biology in Japan. We operate five beamlines for protein crystallography (PX) and two beamlines for biological small-angle X-ray scattering (bioSAXS) at the Photon Factory. Using the PX beamlines and the MR-native SAD method, it is possible to solve the phase problem with the anomalous scattering from sulfur atoms [1]. It is no longer necessary to prepare Se-Met-substituted proteins to solve the protein structures. Within the field of BioSAXS, not only SEC-SAXS but also titration SAXS is possible. The bioSAXS data can also be analyzed using our original software. In addition to the beamlines, a 200kV cryo-EM was installed in 2018 and has been opened for academic and industrial users. SBRC also has a state-of-the-art wet laboratory with an automated crystallization robot [2]. Using these facilities, we have determined the ternary structures of biological macromolecules, including GTP-metabolism relevant molecules and their complexes. In addition to these techniques, it is possible to use hybrid methods in SBRC [3]. Experts in each technique lend support to users of our facilities, offering input and collaboration as needed to help users complete their projects.

To unravel the overall structure of the biological networks, it is not enough to know the relationships between genotypes and phenotypes, because the functional complexities of proteins are substantially more extensive than those of genes. Due to the complex characters of proteins arising from their tertiary structures, dynamics, cellular localizations, interaction partners, and post-translational modifications, it is hardly possible to comprehensively understand their functions in cells from genetic information alone, even in the era of AlphaFold2. In addition, deletion of one protein (or gene) from cells removes several biochemical functions simultaneously, introducing ambiguities into the analysis of results. Such ambiguities hamper precise elucidation of the biological network. To reduce them, researchers have utilized mutant proteins and small molecular weight inhibitors (molecular tools) to probe the biological functions of the target protein(s). However, it is still difficult to regulate their biochemical functions one by one. We have studied GTP metabolism and discovered the first cellular GTP sensor, PI5P4Kβ [4]. Intriguingly, PI5P4Kβ can catalyze kinase reactions using ATP and GTP. Since the binding sites of ATP and GTP are nearly the same, it is difficult to separate the ATP- and GTP-dependent catalytic activities by means of simple mutations. However, separating the two activities was necessary to prove our hypothesis that PI5P4Kβ senses cellular GTP levels and causes cellular responses. To solve this problem, we used high-resolution structural information and designed a PI5P4Kβ mutant protein that only lacks GTP-dependent activity [5]. In this way, we successfully proved our hypothesis. This result demonstrates that fine regulation of the protein functions is possible with the atomic resolution structures. Inhibitors are also critical to analyze protein functions in cells. Since inhibitors can quickly inactivate target proteins, their biological effects are different from those of mutations on cells. In particular, mutant cells frequently show adaptations. One of the critical challenges of the inhibitor design is isotype specificity. Since gene duplication that creates isotypes seems to be an origin of the complexity of life [6], the functional variety of isotypes is an important target for our research. We are currently trying to develop isotype-specific inhibitors for PI5P4Ks. Our group would like to contribute to the creation of molecular tools by revealing the functional tertiary structures of biological macromolecules at atomic resolution. The tertiary structures of the target protein at work will enable us to design a high-performance molecular tool(s) that inhibits a specific biochemical function of the target protein. This type of molecular tool would allow us to unveil the biological function of the target protein. However, it is still hard to reveal downstream signaling/metabolic pathways of the target protein. To complete the map of the biological network, we need to collaborate with other life science researchers such as those in the fields of OMICS, bioinformatics, and molecular evolution. Without these collaborations, structural studies cannot be adequately integrated into biological investigations. However, in the context of such collaboration, the structural study becomes a powerful tool. Our GTP research team is ideally equipped for providing collaboration in integrated biological research and has revealed new biological functions and signaling pathways of the GTP sensor (in preparation). Since GTP is one of the central metabolites in the regulation of cellular activities, we believe that GTP metabolism is relevant to high-level biological phenomena, including behavior and psychological activities. Our structural studies should provide a basis for understanding and regulating high-level biological activities. As a result, our structure analysis team will have exciting opportunities for collaboration. Our current structural targets include PI5P4Ks, IMPDH, and V-ATPase, which are essential players in GTP metabolism. Since we are also trying to understand the evolution of these proteins, structural analyses of target proteins from various species are underway. While the crystal structures of these proteins have already been clarified, we are trying to comprehensively understand the functional structures of these proteins. We can use not only X-ray crystallography but also cryo-EM and bioSAXS to achieve this goal.