Iris D. Young

Structure of photosystem II and substrate binding at room temperature.

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 centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O–O bond formation and O2 evolution. A detailed understanding of the O–O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature 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 resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O–O bond formation mechanisms.

Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II

We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers

X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.

High-speed fixed-target serial virus crystallography

We report a method for serial X-ray crystallography at X-ray free-electron lasers (XFELs), which allows for full use of the current 120-Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallography to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.

DIALS: implementation and evaluation of a new integration package

A new X-ray diffraction data-analysis package is presented with a description of the algorithms and examples of its application to biological and chemical crystallography.

Improving signal strength in serial crystallography with DIALS geometry refinement

For XFEL data, simultaneous refinement of multi-panel detector geometry with thousands of crystal models in the program DIALS improves the integrated signal quality and helps to reduce non-isomorphism

Exploring the dynamic of PSII at room temperature by simultaneous femtosecond X-ray spectroscopy and dffraction

dioxygen, one of nature’s most fascinating and important reactions. The water-splitting reaction takes place at the oxygen evolving complex (OEC), through five intermediate Sstates (S0 to S4), where S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. The structure of PSII in the dark state has been solved by X-ray diffraction and X-ray free electron laser (XFEL) providing information of the structural geometry of the Mn4CaO5 cluster in OEC. However, to fully understand the O-O bond formation mechanism, elucidating the structures of the OEC in the different Sstates is essential. In our recently published study, we report high-resolution structures of PSII at room temperature using XFEL coupling with X-ray spectroscopy under different illumination conditions. This enables us to gain new insights about the dynamic changes in the structure of the Mn4CaO5 cluster as well as the ligands and the bound water molecules. We further use ammonia as water analogue to investigate the water-binding site(s) and discriminate between mechanisms proposed in literature. We discuss the precise role of the water bound to OEC in electron transfer and the water-splitting reaction.

Insights into the oxygen-evolving mechanism of photosynthesis using XFELs

X-ray free electron lasers (XFELs) provide a unique opportunity for time resolved, damage-free studies of dynamic systems due to the ability of femtosecond-long laser pulses of extreme intensity to diffract on a much shorter time scale than the onset of radiation damage. We have applied this technique to the study of water oxidation in photosystem II (PSII), the transmembrane protein where water is converted to oxygen in plants and photosynthetic cyanobacteria. A nanoflow liquid jet1 or acoustic droplet ejection2 is used to deliver microcrystals suspended in buffer to the XFEL beam. Placement of visible lasers in the path of the jet or droplets allows timed illumination for selection of any stable or transient state of the catalytic center in the water-splitting cycle. In recent XFEL experiments, we obtained the first high-resolution room-temperature diffraction data for the dark and twiceilluminated (2F) states of PSII, which allowed us to exclude several proposed mechanisms for water oxidation.3

Improving the models for diffraction used in serial crystallographic data reduction

Serial crystallography is an expanding field at XFELs and at synchrotrons, but serious challenges remain for data processing pipelines. Crystals are typically not rotated in the beam generating ‘still shots’ that represent only a slice of reciprocal space. With fewer reflections to determine unit cell parameters and crystal orientation, and without a direct way to measure mosaic parameters from a rocking curve, predicting which pixels contain signal is difficult. Integration of single pixel reflections at high resolution has required improvements in the accuracy and precision of models used in still shot crystallography, and these improvements are presented here, as implemented in cctbx.xfel and DIALS.

Towards characterization of photo-excited electron transfer and catalysis in natural and artificial systems using XFELs.

The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open capabilities for studying the structure and dynamics of a wide variety of biological and inorganic systems beyond what is possible at synchrotron sources. Although the structure and chemistry at the catalytic sites have been studied intensively in both biological and inorganic systems, a full understanding of the atomic-scale chemistry requires new approaches beyond the steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure at ambient conditions, while overcoming X-ray damage to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by using the intense and ultra-short femtosecond X-ray pulses from an XFEL, where sample is probed before it is damaged. We have developed methodology for simultaneously collecting X-ray diffraction data and X-ray emission spectra, using an energy dispersive spectrometer, at ambient conditions, and used this approach to study the room temperature structure and intermediate states of the photosynthetic water oxidizing metallo-protein, photosystem II. Moreover, we have also used this setup to simultaneously collect the X-ray emission spectra from multiple metals to follow the ultrafast dynamics of light-induced charge transfer between multiple metal sites. A Mn-Ti containing system was studied at an XFEL to demonstrate the efficacy and potential of this method.