Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok’s S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3–7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok’s cycle as high-resolution structures (2.04–2.08 Å). In addition, we report structures of two transient states at 150 and 400 μs, revealing notable structural changes including the binding of one additional ‘water’, Ox, during the S2→S3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S3 state between Ca and Mn1 supports O–O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.

The DIALS project is a collaboration between Diamond Light Source, Lawrence Berkeley National Laboratory and CCP4 to develop a new software suite for the analysis of crystallographic X-ray diffraction data, initially encompassing spot finding, indexing, refinement and integration. The design, core algorithms and structure of the software are introduced, alongside results from the analysis of data from biological and chemical crystallography experiments.

Summary Synaptotagmin-1 and neuronal SNARE proteins play key roles in evoked synchronous neurotransmitter release. However, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca2+- and Mg2+-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many sidechains. The structures revealed several interfaces, including a large, specific, Ca2+-independent, and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca2+-triggered neurotransmitter release in neuronal synapses and for Ca2+-triggered vesicle fusion in a reconstituted system. We propose that this interface forms prior to Ca2+-triggering, and moves en bloc as Ca2+ influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.

Summary The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson’s disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears responsible for amyloid formation and cytotoxicity of α-synuclein. Here we report crystals of NACore having dimensions smaller than the wavelength of visible light and thus invisible by optical microscopy. Thousands of times too small for structure determination by synchrotron x-ray diffraction, these crystals have yielded an atomic resolution structure by the frontier method of Micro-Electron Diffraction. The 1.4 Å resolution structure demonstrates for the first time that this method can determine previously unknown protein structures and here yields the highest resolution achieved by any cryo-electron microscopy method to date. The structure reveals protofibrils built of pairs of face-to-face β-sheets. X-ray fiber diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, opening opportunities for design of inhibitors of α-synuclein fibrils.

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment-protein complex, couples the one-electron photochemistry at the reaction center with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC) (Fig. 1a, Extended Data Fig. 1). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4)1, where S1 is the dark stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution2,3. A detailed understanding of the O-O bond formation mechanism remains a challenge, and elucidating the structures of the OEC in the different S-states, as well as the binding of the two substrate waters to the catalytic site4-6, is a prerequisite for this purpose. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage free, room temperature (RT) structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å structure of PS II7 at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, RT measurements are required to study the structural landscape of proteins under functional conditions8,9, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analog, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states10. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site10-13. Thus, this approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.