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Cytochromecoxidase (CcO) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a hemebgroup in their active sites, CcO has a unique binuclear center (BNC) comprised of a copper atom (CuB) and a hemea3iron, where O2binds and is reduced to water. CO is a versatile O2surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine CcO (bCcO) revealed that photolyzing CO from the hemea3iron leads to a metastable intermediate (CuB-CO), where CO is bound to CuB, before it escapes out of the BNC. Here, with a time-resolved serial femtosecond X-ray crystallography-based pump-probe method, we detected a geminate photoproduct of the bCcO-CO complex, where CO is dissociated from the hemea3iron and moved to a temporary binding site midway between the CuBand the hemea3iron, while the locations of the two metal centers and the conformation of the Helix-X, housing the proximal histidine ligand of the hemea3iron, remain in the CO complex state. This new structure, combined with other reported structures of bCcO, allows the full definition of the ligand dissociation trajectory, as well as the associated protein dynamics.

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macromolecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.

Nature Communications 11(1), 4511 (2020). doi:10.1038/s41467-020-18156-7, Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported., Published by Nature Publishing Group UK, [London]

A pulldown using a biotinylated natural product of interest in the 17β-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), identified the bromodomain and extra-terminal domain (BET) family of proteins (BRD2, BRD3, and BRD4), readers of acetyl-lysine modifications and regulators of gene transcription, as potential cellular targets. BROMOscan bromodomain profiling and biochemical assays support PCC as a BET inhibitor with increased selectivity for bromodomain (BD)-1 of BRD3 and BRD4, and X-ray crystallography and NMR studies uncovered specific contacts that underlie the potency and selectivity of PCC toward BRD3-BD1 over BRD3-BD2. PCC also displays characteristics of a molecular glue, facilitating proteasome-mediated degradation of BRD3 and BRD4. Finally, PCC is more potent than other withanolide analogues and gold-standard pan-BET inhibitor (+)-JQ1 in cytotoxicity assays across five prostate cancer (PC) cell lines regardless of androgen receptor (AR)-signaling status.

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

Nature methods 2020(17), 73–78 (2019). doi:10.1038/s41592-019-0628-z, The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL., Published by Nature Publishing Group, London [u.a.]

Cytochrome c oxidase (C c O) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine C c O. It is assigned to the P R -intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a 3 iron atom is in a ferryl (Fe 4+ = O 2− ) configuration, and heme a and Cu B are oxidized while Cu A is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.

We develop a computational approach to identify distal residues that allosterically modulate the dynamics of binding sites by combining dynamic coupling with statistical analysis of co-evolution. Putative mutants of these predicted allosteric sites are subjected to Adaptive BP-Dock docking tool for binding analysis. Here, we apply this method to a small lectin, Cyanovirin-N (CV-N), that selectively binds to dimannose. Our computational method points out mutations on I34, that is 16A away from binding site can modulate binding. Experimental characterization of I34 mutants confirms that I34Y increases affinity towards dimannose, while I34K completely abolish binding. The increased affinity is not due to changes in the binding region, which are conserved in the crystal structure. However, ITC analysis reveals an opposite contribution of TDS (negative in WT, and positive in I34Y) and suggests that modulation of dynamics (i.e., dynamic allostery) is responsible for the change in binding affinity. Our results point to a novel approach to identify and substitute distal sites, guiding the mutational landscape in glycan-binding proteins to improve binding affinity.

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5’-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds., Trypanosoma brucei inosine-5′-monophosphate dehydrogenase (IMPDH) is an enzyme in the guanine nucleotide biosynthesis pathway and of interest as a drug target. Here the authors present the 2.8 Å room temperature structure of TbIMPDH determined by utilizing X-ray free-electron laser radiation and crystals that were grown in insect cells and find that ATP and GMP are bound at the canonical sites of the Bateman domains.

A homodimeric complex for anaerobic photosynthesis In plants, algae, and cyanobacteria, large molecular complexes—photosystems I and II—convert light energy into chemical energy, releasing oxygen as a by-product. This oxygenic photosynthesis is critical for maintaining Earth's atmospheric oxygen. At their cores, photosystems I and II contain a heterodimeric reaction center. Reaction centers evolved in an atmosphere lacking oxygen, and the ancestral complex was likely homodimeric, encoded by a single gene. Gisriel et al. describe the structure of a homodimeric reaction center from an anoxygenic photosynthetic bacterium. The structure shows perfect symmetry of the light-collecting antennae and elucidates the electron transfer chain. Science , this issue p. 1021

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 A, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 A, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe-C-O angle of ∼142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near CuB, which, in turn, moves closer to the heme a3 iron by ∼0.38 A. Structural comparison reveals that ligand binding to the heme a3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.

Nature Communications 10(1), 11 (2019). doi:10.1038/s41467-019-12955-3, The world’s first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs., Published by Nature Publishing Group UK, [London]

Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the P(R)-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a(3) iron atom is in a ferryl (Fe(4+) = O(2−)) configuration, and heme a and Cu(B) are oxidized while Cu(A) is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.

The journal of physical chemistry letters 10(3), 441 - 446 (2019). doi:10.1021/acs.jpclett.8b03595, X-ray free electron lasers (XFELs) provide ultrashort intense X-ray pulses suitable to probe electron dynamics but can also induce a multitude of nonlinear excitation processes. These affect spectroscopic measurements and interpretation, particularly for upcoming brighter XFELs. Here we identify and discuss the limits to observing classical spectroscopy, where only one photon is absorbed per atom for a Mn$^{2+}$ in a light element (O, C, H) environment. X-ray emission spectroscopy (XES) with different incident photon energies, pulse intensities, and pulse durations is presented. A rate equation model based on sequential ionization and relaxation events is used to calculate populations of multiply ionized states during a single pulse and to explain the observed X-ray induced spectral lines shifts. This model provides easy estimation of spectral shifts, which is essential for experimental designs at XFELs and illustrates that shorter X-ray pulses will not overcome sequential ionization but can reduce electron cascade effects., Published by ACS, Washington, DC

Protein crystallization is a major bottleneck of structure determination by X-ray crystallography, hampering the process by years in some cases. Numerous matrix screening trials using significant amounts of protein are often applied, while a systematic approach with phase diagram determination is prohibited for many proteins that can only be expressed in small amounts. Here, we demonstrate a microfluidic nanowell device implementing protein crystallization and phase diagram screening using nanoscale volumes of protein solution per trial. The device is made with cost-effective materials and is completely automated for efficient and economical experimentation. In the developed device, 170 trials can be realized with unique concentrations of protein and precipitant established by gradient generation and isolated by elastomeric valving for crystallization incubation. Moreover, this device can be further downscaled to smaller nanowell volumes and larger scale integration. The device was calibrated using a fluorescent dye and compared to a numerical model where concentrations of each trial can be quantified to establish crystallization phase diagrams. Using this device, we successfully crystallized lysozyme and C-phycocyanin, as visualized by compatible crystal imaging techniques such as bright-field microscopy, UV fluorescence, and second-order nonlinear imaging of chiral crystals. Concentrations yielding observed crystal formation were quantified and used to determine regions of the crystallization phase space for both proteins. Low sample consumption and compatibility with a variety of proteins and imaging techniques make this device a powerful tool for systematic crystallization studies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fluctuation X-ray scattering (FXS) is an emerging experimental technique in which solution scattering data are collected using X-ray exposures below rotational diffusion times, resulting in angularly anisotropic X-ray snapshots that provide several orders of magnitude more information than traditional solution scattering data. Such experiments can be performed using the ultrashort X-ray pulses provided by a free-electron laser source, allowing one to collect a large number of diffraction patterns in a relatively short time. Here, we describe a test data set for FXS, obtained at the Linac Coherent Light Source, consisting of close to 100 000 multi-particle diffraction patterns originating from approximately 50 to 200 Paramecium Bursaria Chlorella virus particles per snapshot. In addition to the raw data, a selection of high-quality pre-processed diffraction patterns and a reference SAXS profile are provided.

Background Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Results Here, we demonstrate a general method for capturing enzyme catalysis “in action” by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. Conclusions MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data. Electronic supplementary material The online version of this article (10.1186/s12915-018-0524-5) contains supplementary material, which is available to authorized users.

A new approach of using lipidic cubic phase as a carrier matrix for delivering soluble protein microcrystals for serial crystallography helps to dramatically reduce protein consumption. The structures of two soluble test proteins have been determined by this method using less than 0.1 mg of each protein., Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is demonstrated that LCP can also be used as a suitable carrier medium for microcrystals of soluble proteins, enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.

Viscous sample delivery that decreases the net protein consumed in serial femtosecond crystallography is described. The agarose stream has a low background, is compatible with membrane proteins and can be used at a wide range of temperatures., Serial femtosecond crystallography (SFX) has opened a new era in crystallo­graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

SummaryIn proteins, functional divergence involves mutations that modify structure and dynamics. Here we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. Crystallographic structures were determined for several reconstructed ancestral proteins belonging to a GFP class frequently employed in superresolution microscopy. Their chain flexibility was analyzed using molecular dynamics and perturbation response scanning. The green-to-red photoconvertible phenotype appears to have arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the β barrel fold. The allosterically coupled mutational sites provide active site conformational mobility via epistasis. We propose that light-induced chromophore twisting is enhanced in a reverse-protonated subpopulation, activating internal acid-base chemistry and backbone cleavage to enlarge the chromophore. Dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities.

Scientific Data 4:170048 doi: 10.1038/sdata201748 (2017); Published 11 April 2017; Updated 24 October 2017. The Data Descriptor incorrectly states the number of normal incidences used to generate the plot in Fig. 4b as 209. This plot was generated from 32 normal incidence cases.

Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Here, we demonstrate a general method for capturing enzyme catalysis ‘in-action’ by ‘mix-and-inject serial crystallography’. Specifically, we follow the catalytic reaction of theMycobacterium tuberculosisα-lactamase with the 3rdgeneration antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation on the millisecond to second time scales including the crossover from transition state kinetics to steady-state kinetics.SynopsisAn enzymatically catalyzed reaction is initiated by diffusion based mixing of substrate and followed at runtime by time-resolved serial crystallography using a free electron laser.

Scientific data 4, 170048 (2017). doi:10.1038/sdata.2017.48, X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research., Published by Springer Nature, London

Proceedings of the National Academy of Sciences of the United States of America 114(9), 2247 – 2252 (2017). doi:10.1073/pnas.1609243114, To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm$^3$ in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm$^3$ in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach., Published by National Acad. of Sciences, Washington, DC

Biophysical journal 112(3), 506a - 507a (2017). doi:10.1016/j.bpj.2016.11.2740, Published by Cell Press, Cambridge, Mass.

Nature 530(7589), 202 - 206(2016). doi:10.1038/nature16949, The three-dimensional structures of macromolecules and their complexes are mainly elucidated by X-ray protein crystallography. A major limitation of this method is access to high-quality crystals, which is necessary to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields information of sufficiently high resolution with which to solve the crystal structure. The observation that crystals with reduced unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks suggests that crystallographic resolution for some macromolecules may be limited not by their heterogeneity, but by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern that is equal to the incoherent sum of diffraction from rigid individual molecular complexes aligned along several discrete crystallographic orientations and that, consequently, contains more information than Bragg peaks alone. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins—they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5 $\mathring{A}$ limit of measurable Bragg peaks, which allows us to phase the pattern directly. Using the molecular envelope conventionally determined at 4.5 $\mathring{A}$ as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 $\mathring{A}$. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography, using a method that exploits commonly encountered imperfect crystals and enables model-free phasing., Published by Nature Publ. Group, London [u.a.]

Many biological processes depend on detecting and responding to light. The response is often mediated by a structural change in a protein that begins when absorption of a photon causes isomerization of a chromophore bound to the protein. Pande et al. used x-ray pulses emitted by a free electron laser source to conduct time-resolved serial femtosecond crystallography in the time range of 100 fs to 3 ms. This allowed for the real-time tracking of the trans-cis isomerization of the chromophore in photoactive yellow protein and the associated structural changes in the protein.Science, this issue p. 725A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes after photon absorption. The initial step is often the photoisomerization of a conjugated chromophore. Isomerization occurs on ultrafast time scales and is substantially influenced by the chromophore environment. Here we identify structural changes associated with the earliest steps in the trans-to-cis isomerization of the chromophore in photoactive yellow protein. Femtosecond hard x-ray pulses emitted by the Linac Coherent Light Source were used to conduct time-resolved serial femtosecond crystallography on photoactive yellow protein microcrystals over a time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction.

Photosystem II (PSII) is a membrane protein supercomplex that executes the initial reaction of photosynthesis in higher plants, algae, and cyanobacteria. It captures the light from the sun to catalyze a transmembrane charge separation. In a series of four charge separation events, utilizing the energy from four photons, PSII oxidizes two water molecules to obtain dioxygen, four protons, and four electrons. The light reactions of photosystems I and II (PSI and PSII) result in the formation of an electrochemical transmembrane proton gradient that is used for the production of ATP. Electrons that are subsequently transferred from PSI via the soluble protein ferredoxin to ferredoxin-NADP(+) reductase that reduces NADP(+) to NADPH. The products of photosynthesis and the elemental oxygen evolved sustain all higher life on Earth. All oxygen in the atmosphere is produced by the oxygen-evolving complex in PSII, a process that changed our planet from an anoxygenic to an oxygenic atmosphere 2.5 billion years ago. In this chapter, we provide recent insight into the mechanisms of this process and methods used in probing this question.

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution., Diacylglycerol kinase is a small bacterial membrane-bound trimer that catalyses diacylglycerol conversion to phosphatidic acid. Here, the authors solve the crystal structure of the kinase bound to a lipid substrate and an ATP analogue, and show that the active site arose through convergent evolution.

Nature structural & molecular biology 22(3), 265 - 268(2015). doi:10.1038/nsmb.2965, Bifunctional μ- and ​δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human ​δ-OR bound to the bifunctional ​δ-OR antagonist and ​μ-OR agonist tetrapeptide ​H-Dmt-Tic-Phe-Phe-NH2 (​DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between ​H-Dmt and ​Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics., Published by Nature Publishing Group, London [u.a.]

Serial femtosecond crystallography using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomolecules. 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.

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.