Native structure of photosystem II at 1.95 Å resolution viewed by femtosecond X-ray pulses

Photosynthesis converts light energy into biologically useful chemical energy vital to life on Earth. The initial reaction of photosynthesis takes place in photosystem II (PSII), a 700-kilodalton homodimeric membrane protein complex which catalyses photo-oxidation 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 Mn4CaO5-cluster coordinated by a well-defined protein environment1. However, extended X-ray absorption fine structure (EXAFS) studies showed that the manganese cations in the OEC are easily reduced by X-ray irradiation2, and slight differences were found in the Mn–Mn distances between the results of XRD1, EXAFS3–7 and theoretical studies8–14. Here we report a ‘radiation-damage-free’ structure of PSII from Thermosynechococcus vulcanus in the S1 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 laser structure has Mn–Mn distances that are shorter by 0.1–0.2 Å. The valences of each manganese atom were tentatively assigned as Mn1D(III), Mn2C(IV), Mn3B(IV) and Mn4A(III), based on the average Mn–ligand distances and analysis of the Jahn–Teller axis on Mn(III). One of the oxo-bridged oxygens, O5, has significantly longer Mn–O distances in contrast to the other oxo-oxygen atoms, suggesting that it is a hydroxide ion instead of a normal oxygen dianion and therefore may serve as one of the substrate oxygen atoms. 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

Radioprotectant screening for cryocrystallography.

Radiation damage continues to present a problem to crystallographers using cryocooled protein crystals at third-generation synchrotrons. Free-radical scavengers have been suggested as a possible means of reducing the rate of this damage. The screening of a large number of potential radioprotectants was undertaken with an online microspectrophotometer using cystine and cysteine to model protein disulfide bonds and thiol groups, respectively. Oxidized alpha-lipoic acid was tested as a possible model disulfide bond. The evidence for the effectiveness of ascorbate as a radioprotectant was strengthened, and quinone, 2,2,6,6-tetramethyl-4-piperidone, and reduced dithiothreitol showed promise as radioprotectants

Observation of decreased radiation damage at higher dose rates in room temperature protein crystallography.

Radiation damage can be a problem when utilizing ionizing X-radiation in macromolecular crystallography. The dose dependence of radiation damage to eight lysozyme crystals at room temperature (292 K) was investigated in order to provide an accurate comparison with cryotemperature (100 K) results and to allow researchers to calculate expected maximum room-temperature-crystal lifetimes prior to data collection. Results of intensity-loss analysis unexpectedly showed that the dose tolerated by a crystal is dependent on the dose rate according to a positive linear relationship (99% correlation coefficient); a 60% increase in dose rate gave a 4-fold increase in crystal lifetime over the range studied. Alternative metrics of damage were also assessed from room temperature data. In the dose-rate range tested (6 Gy s(-1) to 10 Gy s(-1)), data collection at 100 K appears to offer a 26-113 times increase in the lifetime of the crystal

Room-temperature scavengers for macromolecular crystallography: increased lifetimes and modified dose dependence of the intensity decay.

The advent of highly intense wiggler and undulator beamlines has reintroduced the problem of X-ray radiation damage in protein crystals even at cryogenic temperatures (100 K). Although cryocrystallography can be utilized for the majority of protein crystals, certain macromolecular crystals (e.g. of viruses) suffer large increases in mosaicity upon flash cooling and data are still collected at room temperature (293 K). An alternative mechanism to cryocooling for prolonging crystal lifetime is the use of radioprotectants. These compounds are able to scavenge the free radical species formed upon X-ray irradiation which are thought to be responsible for part of the observed damage. Three putative radioprotectants, ascorbate, 1,4-benzoquinone and 2,2,6,6-tetramethyl-4-piperidone (TEMP), were tested for their ability to prolong lysozyme crystal lifetimes at 293 K. Plots of relative summed intensity against dose were used as a metric to assess radioprotectant ability: ascorbate and 1,4-benzoquinone appear to be effective, whereas studies on TEMP were inconclusive. Ascorbate, which scavenges OH* radicals (k(OH) = 8 x 10(9) M(-1) s(-1)) and electrons with a lower rate constant (k(e-(aq)) = 3.0 x 10(8) M(-1) s(-1)), doubled the crystal dose tolerance, whereas 1,4-benzoquinone, which also scavenges both OH* radicals (k(OH) = 1.2 x 10(9) M(-1) s(-1)) and electrons (k(e-(aq)) = 1.2 x 10(10) M(-1) s(-1)), offered a ninefold increase in dose tolerance at the dose rates used. Pivotally, these preliminary results on a limited number of samples show that the two scavengers also induced a striking change in the dose dependence of the intensity decay from a first-order to a zeroth-order process

Practical radiation damage-induced phasing

International audienceAlthough crystallographers typically seek to mitigate radiation damage in macromolecular crystals, in some cases, radiation damage to specific atoms can be used to determine phases de novo. This process is called radiation damage-induced phasing or "RIP." Here, we provide a general overview of the method and a practical set of data collection and processing strategies for phasing macromolecular structures using RIP