of water into dioxygen through an S-state cycle of the oxygen evolving complex (OEC).The structure of PSII has been solved by X-ray diffraction (XRD) at 1.9-ångström (Å) resolution, which revealed that the OEC is a Mn 4 CaO 5 -cluster coordinated by a well-defined protein environment 1 . However, extended X-ray absorption fine structure (EXAFS) studies showed that the manganese cations in the OEC are easily reduced by X-ray irradiation 2 , and slight differences were found in the Mn-Mn distances between the results of XRD 1 , EXAFS 3-7 and theoretical studies 8-14 . Here we report a 'radiation-damage-free' structure of PSII from Thermosynechococcus vulcanus in the S 1 state at a resolution of 1.95 Å using femtosecond X-ray pulses of the SPring-8 ångström compact free-electron laser (SACLA) and a huge number of large, highly isomorphous PSII crystals. Compared with the structure from XRD, the OEC in the X-ray free electron These findings provide a structural basis for the mechanism of oxygen evolution, and we expect that this structure will provide a blueprint for design of artificial catalysts for water oxidation.PSII is a multi-subunit pigment-protein complex embedded in the thylakoid membranes of higher plants, green algae and cyanobacteria, and is the only molecular machine capable of oxidizing water by use of visible light. Water molecules are split into electrons, hydrogen atoms and oxygen molecules at the catalytic centre of PSII, namely, the OEC, through four electron and/or proton removing steps as described in the S i -state cycle (with i = 0-4, where i indicates the number of oxidative equivalents accumulated). Because of its ability to split water, the OEC is considered a promising template for the synthesis of artificial catalysts for water-splitting aimed at obtaining clean and renewable energy from sunlight, which is considered a promising way to supplement the consumption of limited and environmentally unfriendly fossil fuels.In order to elucidate the mechanism of the water-splitting reaction, the structure of PSII has been studied extensively by XRD, with a resolution that has gradually increased from 3.8 to 1.9 Å using synchrotron radiation (SR) X-ray sources 1,[15][16][17][18] . In particular, the SR structure of PSII at atomic resolution revealed that the OEC is a Mn 4 CaO 5 cluster organized into a distorted-chair shape, in which the cuboidal part is composed of Mn 3 CaO 3 and the outer manganese is attached via two -oxo-bridges 1 . The high-resolution structure also revealed that four water molecules are coordinated to the Mn 4 CaO 5 cluster, among which, two are coordinated and XRD studies 1 ). Although these provided an important structural basis for the mechanism of water-splitting, the average Mn-ligand and Mn-Mn distances were found to be slightly longer than those deduced from EXAFS 3-7 and from computational analysis based on the SR structure [8][9][10][11][12][13][14] . This has been suggested to result from radiation damage, as hydrated electrons generated by X-ray irradiation 19 a...

Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S0 to S4). To reveal the mechanism of water oxidation, we analyzed structures of PSII in the S1, S2, and S3 states by x-ray free-electron laser serial crystallography. No insertion of water was found in S2, but flipping of D1 Glu189 upon transition to S3 leads to the opening of a water channel and provides a space for incorporation of an additional oxygen ligand, resulting in an open cubane Mn4CaO6 cluster with an oxyl/oxo bridge. Structural changes of PSII between the different S states reveal cooperative action of substrate water access, proton release, and dioxygen formation in photosynthetic water oxidation.

Norovirus 3C-like proteases are crucial to proteolytic processing of norovirus polyproteins. We determined the crystal structure of the 3C-like protease from Chiba virus, a norovirus, at 2.8-Å resolution. An active site including Cys139 and His30 is present, as is a hydrogen bond network that stabilizes the active site conformation. In the oxyanion hole backbone, a structural difference was observed probably upon substrate binding. A peptide substrate/enzyme model shows that several interactions between the two components are critical for substrate binding and that the S1 and S2 sites appropriately accommodate the substrate P1 and P2 residues, respectively. Knowledge of the structure and a previous mutagenesis study allow us to correlate proteolysis and structure.

Owing to the development of brilliant microfocus beamlines, rapid-readout detectors and sample changers, protein microcrystallography is rapidly becoming a popular technique for accessing structural information from complex biological samples. However, the method is time-consuming and labor-intensive and requires technical expertise to obtain high-resolution protein crystal structures. At SPring-8, an automated data-collection system named ZOO has been developed. This system enables faster data collection, facilitates advanced data-collection and data-processing techniques, and permits the collection of higher quality data. In this paper, the key features of the functionality put in place on the SPring-8 microbeam beamline BL32XU are described and the major advantages of this system are outlined. The ZOO system will be a major driving force in the evolution of the macromolecular crystallography beamlines at SPring-8.

Time-resolved serial femtosecond crystallography using an X-ray free electron laser (XFEL) in conjunction with a photosensitive caged-compound offers a crystallographic method to track enzymatic reactions. Here we demonstrate the application of this method using fungal NO reductase, a heme-containing enzyme, at room temperature. Twenty milliseconds after caged-NO photolysis, we identify a NO-bound form of the enzyme, which is an initial intermediate with a slightly bent Fe-N-O coordination geometry at a resolution of 2.1 Å. The NO geometry is compatible with those analyzed by XFEL-based cryo-crystallography and QM/MM calculations, indicating that we obtain an intact Fe3+-NO coordination structure that is free of X-ray radiation damage. The slightly bent NO geometry is appropriate to prevent immediate NO dissociation and thus accept H− from NADH. The combination of using XFEL and a caged-compound is a powerful tool for determining functional enzyme structures during catalytic reactions at the atomic level.