Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC)^1,2,3. The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn₄CaO₅ cluster organized in an asymmetric, ‘distorted-chair’ form⁴. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the ‘radiation damage-free’⁵ structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the Q_B/non-haem iron and the Mn₄CaO₅ cluster. The changes around the Q_B/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn₄CaO₅ cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ₄-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously^6,7.

Hard X-rays have exceptional properties that are useful in the chemical, elemental and structure analysis of matter. Although single-nanometre resolutions in various hard-X-ray analytical methods are theoretically possible with a focused hard-X-ray beam, fabrication of the focusing optics remains the main hurdle. Aberrations owing to imperfections in the optical system degrade the quality of the focused beam¹. Here, we describe an in situ wavefront-correction approach to overcome this and demonstrate an X-ray beam focused in one direction to a width of 7 nm at 20 keV. We achieved focal spot improvement of the X-ray nanobeam produced by a laterally graded multilayer mirror². A grazing-incidence deformable mirror³ was used to restore the wavefront shape. Using this system, ideal focusing conditions are achievable even if hard-X-ray focusing elements do not achieve sufficient performance. It is believed that this will ultimately lead to single-nanometre spatial resolution in X-ray analytical methods.

Snapshots of bacteriorhodopsin: Bacteriorhodopsin is a membrane protein that harvests the energy content from light to transport protons out of the cell against a transmembrane potential. Nango et al. used timeresolved serial femtosecond crystallography at an x-ray free electron laser to provide 13 structural snapshots of the conformational changes that occur in the nanoseconds to milliseconds after photoactivation. These changes begin at the active site, propagate toward the extracellular side of the protein, and mediate internal protonation exchanges that achieve proton transport. Science , this issue p. 1552

Inspecting S states in photosynthesis: Oxygenic photosynthesis uses a Mn ₄ CaO ₅ cluster in the oxygen-evolving complex to extract electrons from water and produce dioxygen. Visualizing each of the chemical states in this process, S ₀ to S ₄ , and assigning chemical identities and mechanisms on the basis of structures has been a challenge addressed recently by work at x-ray free-electron lasers. Suga et al. used serial crystallography at cryogenic temperatures to trap and determine the structures of several stable states during photosystem II water oxidation (see the Perspective by Britt and Marchiori). Changes around the water cluster already happen in the S ₂ state and set the stage for water insertion that occurs during transition to the S ₃ state. A short 1.9-angstrom distance between the two oxygen atoms in the S ₃ state is consistent with theoretical studies supporting an oxyl/oxo mechanism for oxygen-oxygen coupling. Science , this issue p. 334 ; see also p. 305