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The hemoprotein myoglobin is a model system for the study of protein dynamics. We used time-resolved serial femtosecond crystallography at an x-ray free-electron laser to resolve the ultrafast structural changes in the carbonmonoxy myoglobin complex upon photolysis of the Fe-CO bond. Structural changes appear throughout the protein within 500 femtoseconds, with the C, F, and H helices moving away from the heme cofactor and the E and A helices moving toward it. These collective movements are predicted by hybrid quantum mechanics/molecular mechanics simulations. Together with the observed oscillations of residues contacting the heme, our calculations support the prediction that an immediate collective response of the protein occurs upon ligand dissociation, as a result of heme vibrational modes coupling to global modes of the protein.

Serial femtosecond crystallography using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomolecuies. We used microcrystals of photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resolution, time-resolved difference electron density maps of excellent quality with strong features; these allowed the determination of structures of reaction intermediates to a resolution of 1.6 angstroms. Our results open the way to the study of reversible and nonreversible biological reactions on time scales as short as femtoseconds under conditions that maximize the extent of reaction initiation throughout the crystal.

Intense femtosecond x-ray pulses produced at the Linac Coherent Light Source (LCLS) were used for simultaneous x-ray diffraction (XRD) and x-ray emission spectroscopy (XES) of microcrystals of photosystem II (PS II) at room temperature. This method probes the overall protein structure and the electronic structure of the Mn₄ CaO₅ cluster in the oxygen-evolving complex of PS II. XRD data are presented from both the dark state (S₁) and the first illuminated state (S₂) of PS II. Our simultaneous XRD-XES study shows that the PS II crystals are intact during our measurements at the LCLS, not only with respect to the structure of PS II, but also with regard to the electronic structure of the highly radiation-sensitive Mn₄CaO₅ cluster, opening new directions for future dynamics studies.

The dioxygen we breathe is formed by light-induced oxidation of water in photosystem II. O 2 formation takes place at a catalytic manganese cluster within milliseconds after the photosystem II reaction centre is excited by three single-turnover flashes. Here we present combined X-ray emission spectra and diffraction data of 2-flash (2F) and 3-flash (3F) photosystem II samples, and of a transient 3F’ state (250 μs after the third flash), collected under functional conditions using an X-ray free electron laser. The spectra show that the initial O–O bond formation, coupled to Mn reduction, does not yet occur within 250 μs after the third flash. Diffraction data of all states studied exhibit an anomalous scattering signal from Mn but show no significant structural changes at the present resolution of 4.5 Å. This study represents the initial frames in a molecular movie of the structural changes during the catalytic reaction in photosystem II. Photosystem II is the biosynthetic machinery that allows the conversion of water to oxygen using light. Here, the authors combine X-ray emission and diffraction data to probe the structural changes that take place during photosystem II catalysis.

Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX. Serial femtosecond crystallography using X-ray free-electron lasers has huge potential for time-resolved structural experiments. Here, the authors present a structure of the light-driven proton pump bacteriorhodopsin using these techniques.

Femtosecond X-ray pulses were used to obtain diffraction data on photosystem II, revealing conformational changes as the complex transitions from the dark S 1 state to the double-pumped S 3 state; the time-resolved serial femtosecond crystallography technique enables structural determination of protein conformations that are highly prone to traditional radiation damage. An X-ray snapshot of photosystem II structure It has recently been shown that extremely short and intense radiation pulses from X-ray free-electron lasers can be used to obtain diffraction data on nanometre- to micrometre-sized protein crystals before the crystal suffers radiation damage. The hope is that this 'serial femtosecond crystallography' (SFX) approach will produce structures of proteins and protein complexes that do not yield well-ordered macroscopic crystals. These authors collected time-resolved SFX data on small crystals of photosystem II of photosynthesis during its transition from the 'dark' S1 state to the double-excited S3 state. At present the resolution of this technique is moderate, but it is sufficient to reveal significant conformational changes at the Mn 4 CaO 5 cluster at the heart of the oxygen evolving complex and at the electron acceptor site. Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth’s oxygenic atmosphere 1 . In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S 0 to S 4 , in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed 2 technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S 1 state and after double laser excitation (putative S 3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn 4 CaO 5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the ‘dangler’ Mn) and the Mn 3 CaO x cubane in the S 2 to S 3 transition, as predicted by spectroscopic and computational studies 3 , 4 . This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

A 'protein quake' is directly monitored on the picosecond timescale using the method of time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography 1 using an X-ray free-electron laser 2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions. Time-resolved serial femtosecond crystallography is used to reveal the structural changes that stabilize the charge-separation steps of electron-transfer reactions in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds.

Explosions are spectacular and intriguing phenomena that expose the dynamics of matter under extreme conditions. We investigated, using time-resolved imaging, explosions induced by ultraintense X-ray laser pulses in water drops and jets. Our observations revealed an explosive vaporization followed by high-velocity interacting flows of liquid and vapour, and by the generation of shock trains in the liquid jets. These flows are different from those previously observed in laser ablation, owing to a simpler spatial pattern of X-ray absorption. We show that the explosion dynamics in our experiments is consistent with a redistribution of absorbed energy, mediated by a pressure or shock wave in the liquid, and we model the effects of explosions, including their adverse impact on X-ray laser experiments. X-ray laser explosions have predictable dynamics that may prove useful for controlling the state of pure liquids over broad energy scales and timescales, and for triggering pressure-sensitive molecular dynamics in solutions. X-ray-induced explosions in water drops, examined using time-resolved imaging, show interacting high-speed liquid and vapour flows. This type of X-ray absorption dynamics is predictable and may be used for inducing particular dynamical liquid states.