Cytochrome c oxidase (C c O) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a 3 of ba 3 -type C c O. In contrast with the aa 3 -type enzyme, our data show how CO is stabilized on Cu B through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the Cu B -CO complex in ba 3 -type C c O and, by extension, the extremely high oxygen affinity of the enzyme.

Serial crystallography is a rapidly growing method that can yield structural insights from microcrystals that were previously considered to be too small to be useful in conventional X-ray crystallography. Here, conditions for growing microcrystals of the photosynthetic reaction centre of Blastochloris viridis within a lipidic cubic phase (LCP) crystallization matrix that employ a seeding protocol utilizing detergent-grown crystals with a different crystal packing are described. LCP microcrystals diffracted to 2.25 Å resolution when exposed to XFEL radiation, which is an improvement of 0.15 Å over previous microcrystal forms. Ubiquinone was incorporated into the LCP crystallization media and the resulting electron density within the mobile QB pocket is comparable to that of other cofactors within the structure. As such, LCP microcrystallization conditions will facilitate time-resolved diffraction studies of electron-transfer reactions to the mobile quinone, potentially allowing the observation of structural changes associated with the two electron-transfer reactions leading to complete reduction of the ubiquinone ligand.

Time-resolved x-ray solution scattering (TR-XSS) is a sub-field of structural biology, which observes secondary structural changes in proteins as they evolve along their functional pathways. While the number of distinct conformational states and their rise and decay can be extracted directly from TR-XSS experimental data recorded from light-sensitive systems, structural modeling is more challenging. This step often builds from complementary structural information, including secondary structural changes extracted from crystallographic studies or molecular dynamics simulations. When working with integral membrane proteins, another challenge arises because x-ray scattering from the protein and the surrounding detergent micelle interfere and these effects should be considered during structural modeling. Here, we utilize molecular dynamics simulations to explicitly incorporate the x-ray scattering cross term between a membrane protein and its surrounding detergent micelle when modeling TR-XSS data from photoactivated samples of detergent solubilized bacteriorhodopsin. This analysis provides theoretical foundations in support of our earlier approach to structural modeling that did not explicitly incorporate this cross term and improves agreement between experimental data and theoretical predictions at lower x-ray scattering angles.

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.

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 crystallography1 using an X-ray free-electron laser2 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.

Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.

: Ultrafast isomerization of retinal is the primary step in a range of photoresponsive biological functions including vision in humans and ion-transport across bacterial membranes. We studied the sub-picosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin using an X-ray laser. Twenty snapshots with near-atomic spatial and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket prior to passing through a highly-twisted geometry and emerging in the 13-cis conformation. The aspartic acid residues and functional water molecules in proximity of the retinal Schiff base respond collectively to formation and decay of the initial excited state and retinal isomerization. These observations reveal how the protein scaffold guides this remarkably efficient photochemical reaction.

Cytochrome c oxidase catalyses the reduction of molecular oxygen to water while the energy released in this process is used to pump protons across a biological membrane. Although an extremely well-studied biological system, the molecular mechanism of proton pumping by cytochrome c oxidase is still not understood. Here we report a method to produce large quantities of highly diffracting microcrystals of ba 3-type cytochrome c oxidase from Thermus thermophilus suitable for serial femtosecond crystallography. The room-temperature structure of cytochrome c oxidase is solved to 2.3 Å resolution from data collected at an X-ray Free Electron Laser. We find overall agreement with earlier X-ray structures solved from diffraction data collected at cryogenic temperature. Previous structures solved from synchrotron radiation data, however, have shown conflicting results regarding the identity of the active-site ligand. Our room-temperature structure, which is free from the effects of radiation damage, reveals that a single-oxygen species in the form of a water molecule or hydroxide ion is bound in the active site. Structural differences between the ba 3-type and aa 3-type cytochrome c oxidases around the proton-loading site are also described.

Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RCvir). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RCvir serial femtosecond crystallography (SFX) structure from 3.3-Å to 2.4-Å resolution. This approach may allow known crystallization conditions for other proteins to be adapted to exploit novel scientific opportunities created by serial crystallography.