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...

Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii) at 2.3 Å resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties.

The major facilitator superfamily glucose transporters, exemplified by human GLUT1-4, have been central to the study of solute transport. Using lipidic cubic phase crystallization and microfocus X-ray diffraction, we determined the structure of human GLUT3 in complex with D-glucose at 1.5 Å resolution in an outward-occluded conformation. The high-resolution structure allows discrimination of both α- and β-anomers of D-glucose. Two additional structures of GLUT3 bound to the exofacial inhibitor maltose were obtained at 2.6 Å in the outward-open and 2.4 Å in the outward-occluded states. In all three structures, the ligands are predominantly coordinated by polar residues from the carboxy terminal domain. Conformational transition from outward-open to outward-occluded entails a prominent local rearrangement of the extracellular part of transmembrane segment TM7. Comparison of the outward-facing GLUT3 structures with the inward-open GLUT1 provides insights into the alternating access cycle for GLUTs, whereby the C-terminal domain provides the primary substrate-binding site and the amino-terminal domain undergoes rigid-body rotation with respect to the C-terminal domain. Our studies provide an important framework for the mechanistic and kinetic understanding of GLUTs and shed light on structure-guided ligand design.

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.