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Inorganic–organic hybrid materials represent a large share of newly reported structures, owing to their simple synthetic routes and customizable properties1. This proliferation has led to a characterization bottleneck: many hybrid materials are obligate microcrystals with low symmetry and severe radiation sensitivity, interfering with the standard techniques of single-crystal X-ray diffraction2,3 and electron microdiffraction4–11. Here we demonstrate small-molecule serial femtosecond X-ray crystallography (smSFX) for the determination of material crystal structures from microcrystals. We subjected microcrystalline suspensions to X-ray free-electron laser radiation12,13 and obtained thousands of randomly oriented diffraction patterns. We determined unit cells by aggregating spot-finding results into high-resolution powder diffractograms. After indexing the sparse serial patterns by a graph theory approach14, the resulting datasets can be solved and refined using standard tools for single-crystal diffraction data15–17. We describe the ab initio structure solutions of mithrene (AgSePh)18–20, thiorene (AgSPh) and tethrene (AgTePh), of which the latter two were previously unknown structures. In thiorene, we identify a geometric change in the silver–silver bonding network that is linked to its divergent optoelectronic properties20. We demonstrate that smSFX can be applied as a general technique for structure determination of beam-sensitive microcrystalline materials at near-ambient temperature and pressure., Small-molecule serial femtosecond X-ray crystallography (smSFX) characterizes microcrystals by indexing sparse serial XFEL diffraction frames, with little sample preparation, without beam damage, and at room temperature and pressure.

The mechanisms by which enzymes promote catalytic reactions efficiently through their structural changes remain to be fully elucidated. Recent progress in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers (XFELs) has made it possible to address these issues. In particular, mix-and-inject serial crystallography (MISC) is promising for the direct observation of structural changes associated with ongoing enzymic reactions. In this study, SFX measurements using a liquid-jet system were performed on microcrystals of bacterial copper amine oxidase anaerobically premixed with a substrate amine solution. The structure determined at 1.94 Å resolution indicated that the peptidyl quinone cofactor is in equilibrium between the aminoresorcinol and semiquinone radical intermediates, which accumulate only under anaerobic single-turnover conditions. These results show that anaerobic conditions were well maintained throughout the liquid-jet SFX measurements, preventing the catalytic intermediates from reacting with dioxygen. These results also provide a necessary framework for performing time-resolved MISC to study enzymic reaction mechanisms under anaerobic conditions.

Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to trigger conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the sub-millisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature-jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.

International audience; Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD•- isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge allows proton transfer from arginine to FAD•-. Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.

A method for determining the sample flow rate and concomitant light condition in time-resolved serial femtosecond crystallography is developed to analyze the intermediate-state structures of photosystem II., Photosystem II (PSII) catalyzes light-induced water oxidation through an Si-state cycle, leading to the generation of di-oxygen, protons and electrons. Pump–probe time-resolved serial femtosecond crystallography (TR-SFX) has been used to capture structural dynamics of light-sensitive proteins. In this approach, it is crucial to avoid light contamination in the samples when analyzing a particular reaction intermediate. Here, a method for determining a condition that avoids light contamination of the PSII microcrystals while minimizing sample consumption in TR-SFX is described. By swapping the pump and probe pulses with a very short delay between them, the structural changes that occur during the S1-to-S2 transition were examined and a boundary of the excitation region was accurately determined. With the sample flow rate and concomitant illumination conditions determined, the S2-state structure of PSII could be analyzed at room temperature, revealing the structural changes that occur during the S1-to-S2 transition at ambient temperature. Though the structure of the manganese cluster was similar to previous studies, the behaviors of the water molecules in the two channels (O1 and O4 channels) were found to be different. By comparing with the previous studies performed at low temperature or with a different delay time, the possible channels for water inlet and structural changes important for the water-splitting reaction were revealed.

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3., 光でイオンを輸送する膜タンパク質の巧妙な仕組み --XFELが捉えた光駆動型イオンポンプロドプシンの構造変化--. 京都大学プレスリリース. 2022-02-28.

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data ofNonlabens marinusrhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10 μs and 1 ms time-points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied with a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.SignificanceLight-driven chloride pumps have been identified in various species, including archaea and marine flavobacteria. The function of ion transportation controllable by light is utilized for optogenetics tools in neuroscience. Chloride pumps differ among species, in terms of amino acid homology and structural similarity. Our time-resolved crystallographic studies using X-ray free electron lasers reveal the molecular mechanism of halide ion transfer in a light-driven chloride pump from a marine flavobacterium. Our data indicate a common mechanism in chloride pumping rhodopsins, as compared to previous low temperature trapping studies of chloride pumps. These findings are significant not only for further improvements of optogenetic tools but also for a general understanding of the ion pumping mechanisms of microbial rhodopsins.

Recent advances in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers have paved the way for determining radiation-damage-free protein structures under nonfreezing conditions. However, the large-scale preparation of high-quality microcrystals of uniform size is a prerequisite for SFX, and this has been a barrier to its widespread application. Here, a convenient method for preparing high-quality microcrystals of a bacterial quinoprotein enzyme, copper amine oxidase from Arthrobacter globiformis, is reported. The method consists of the mechanical crushing of large crystals (5–15 mm3), seeding the crushed crystals into the enzyme solution and standing for 1 h at an ambient temperature of ∼26°C, leading to the rapid formation of microcrystals with a uniform size of 3–5 µm. The microcrystals diffracted X-rays to a resolution beyond 2.0 Å in SFX measurements at the SPring-8 Angstrom Compact Free Electron Laser facility. The damage-free structure determined at 2.2 Å resolution was essentially identical to that determined previously by cryogenic crystallography using synchrotron X-ray radiation.

A high-viscosity cartridge-type injector for serial femtosecond crystallography has been developed at SPring-8 Angstrom Compact Free-Electron Laser., A sample-injection device has been developed at SPring-8 Angstrom Compact Free-Electron Laser (SACLA) for serial femtosecond crystallography (SFX) at atmospheric pressure. Microcrystals embedded in a highly viscous carrier are stably delivered from a capillary nozzle with the aid of a coaxial gas flow and a suction device. The cartridge-type sample reservoir is easily replaceable and facilitates sample reloading or exchange. The reservoir is positioned in a cooling jacket with a temperature-regulated water flow, which is useful to prevent drastic changes in the sample temperature during data collection. This work demonstrates that the injector successfully worked in SFX of the human A2A adenosine receptor complexed with an antagonist, ZM241385, in lipidic cubic phase and for hen egg-white lysozyme microcrystals in a grease carrier. The injection device has also been applied to many kinds of proteins, not only for static structural analyses but also for dynamics studies using pump–probe techniques.

One of the sweetest proteins found in tropical fruits (with a threshold of 50 nM), thaumatin, is also used commercially as a sweetener. Our previous study successfully produced the sweetest thaumatin mutant (D21N), designated hyper-sweet thaumatin, which decreases the sweetness threshold to 31 nM. To investigate why the D21N mutant is sweeter than wild-type thaumatin, we compared the structure of the D21N mutant solved at a subatomic resolution of 0.93 A with that of wild-type thaumatin determined at 0.90 A. Although the overall structure of the D21N mutant resembles that of wild-type thaumatin, our subatomic resolution analysis successfully assigned and discriminated the detailed atomic positions of side-chains at position 21. The relative B-factor value of the side-chain at position 21 in the D21N mutant was higher than that of wild-type thaumatin, hinting at a greater flexibility of side-chain at 21 in the hyper-sweet D21N mutant. Furthermore, alternative conformations of Lys19, which is hydrogen-bonded to Asp21 in wild-type, were found only in the D21N mutant. Subatomic resolution analysis revealed that flexible conformations at the sites adjacent to positions 19 and 21 play a crucial role in enhancing sweet potency and may serve to enhance the complementarity of electrostatic potentials for interaction with the sweet taste receptor.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore., X線自由電子レーザーを用いて、光照射によるチャネルロドプシンの構造変化の過程を捉えることに成功. 京都大学プレスリリース. 2021-03-26.

Photosystem II (PSII) catalyzes light-induced water oxidation through an Si-state cycle, leading to the generation of di-oxygen, protons, and electrons. Pump-probe time-resolved serial femtosecond crystallography (TR-SFX) has been used to capture intermediate states of light-driven enzymatic reactions. In this approach, it is crucial to avoid contamination of light into the samples when analyzing a particular reaction intermediate. Here, we describe a method for determining a proper light condition that avoids light contamination to the PSII microcrystals while minimizing the sample consumption in TR-SFX. With the proper illumination conditions determined, we analyzed the S2-state structure of PSII at room temperature, revealing the structural changes during the S1-to-S2 transition at an ambient temperature. By comparing with the previous studies performed at a low temperature or with a different delay time, we reveal the possible channels for water inlet and proton egress, as well as structural changes important for the water-splitting reaction.

Serial femtosecond crystallography (SFX) has enabled determination of room temperature structures of proteins with minimum radiation damage. A highly viscous grease matrix acting as a crystal carrier for serial sample loading at a low flow rate of ~0.5 μl min−1 was introduced into the beam path of X-ray free-electron laser. This matrix makes it possible to determine the protein structure with a sample consumption of less than 1 mg of the protein. The viscosity of the matrix is an important factor in maintaining a continuous and stable sample column from a nozzle of a high viscosity micro-extrusion injector for serial sample loading. Using conventional commercial grease (an oil-based, viscous agent) with insufficient control of viscosity in a matrix often gives an unexpectedly low viscosity, providing an unstable sample stream, with effects such as curling of the stream. Adjustment of the grease viscosity is extremely difficult since the commercial grease contains unknown compounds, which may act as unexpected inhibitors of proteins. This study introduces two novel grease matrix carriers comprising known compounds with a viscosity higher than that of conventional greases, to determine the proteinase K structure from nano-/microcrystals.

The applicability of the ligand-soaking method in serial femtosecond crystallography has been examined to examine the feasibility of pharmaceutical applications of X-ray free-electron lasers., Serial femtosecond crystallography (SFX) with an X-ray free-electron laser is used for the structural determination of proteins from a large number of microcrystals at room temperature. To examine the feasibility of pharmaceutical applications of SFX, a ligand-soaking experiment using thermolysin microcrystals has been performed using SFX. The results were compared with those from a conventional experiment with synchrotron radiation (SR) at 100 K. A protein–ligand complex structure was successfully obtained from an SFX experiment using microcrystals soaked with a small-molecule ligand; both oil-based and water-based crystal carriers gave essentially the same results. In a comparison of the SFX and SR structures, clear differences were observed in the unit-cell parameters, in the alternate conformation of side chains, in the degree of water coordination and in the ligand-binding mode.

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

Proteinase K is widely used in molecular biology for its broad substrate specificity, wide pH stability, and high hydrolysis activity. Aminolysis by proteinase K is also attractive for chemoenzymatic peptide synthesis. However, the relatively low thermal stability of the native protease limits the hydrolysis and aminolysis reactions in industrial processes. Here, we enhanced the thermal stability of proteinase K using a heavy-atom derivatization technique. The denaturation temperature of proteinase K derivatized with praseodymium (Pr) ions was 16.2 and 5.9 °C higher than those of metal-free and Ca-binding proteinase K, respectively. Isothermal titration calorimetry (ITC) measurements demonstrated that Pr-ion binding to proteinase K showed endothermic peaks, whereas Ca-ion binding showed exothermic peaks, indicating that the binding mode of Pr ions was different from that of Ca ions, even though the crystal structures of proteinase K with Pr and Ca ions were identical. Hydrolytic activity of Pr-derivatized...

Serial femtosecond crystallography (SFX) has enabled the damage-free structural determination of metalloenzymes and filled the gaps of our knowledge between crystallographic and spectroscopic data. Crystallographers, however, scarcely know whether the rising technique provides truly new structural insights into mechanisms of metalloenzymes partly because of limited resolutions. Copper nitrite reductase (CuNiR), which converts nitrite to nitric oxide in denitrification, has been extensively studied by synchrotron radiation crystallography (SRX). Although catalytic Cu (Type 2 copper (T2Cu)) of CuNiR had been suspected to tolerate X-ray photoreduction, we here showed that T2Cu in the form free of nitrite is reduced and changes its coordination structure in SRX. Moreover, we determined the completely oxidized CuNiR structure at 1.43 Å resolution with SFX. Comparison between the high-resolution SFX and SRX data revealed the subtle structural change of a catalytic His residue by X-ray photoreduction. This finding, which SRX has failed to uncover, provides new insight into the reaction mechanism of CuNiR. published

Sulfur SAD phasing facilitates the structure determination of diverse native proteins using femtosecond X-rays from free-electron lasers via serial femtosecond crystallography., Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Å is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

An experimental platform for serial femtosecond crystallography using an X-ray free-electron laser and its applications at SACLA are described., An experimental system for serial femtosecond crystallography using an X-ray free-electron laser (XFEL) has been developed. It basically consists of a sample chamber, fluid injectors and a two-dimensional detector. The chamber and the injectors are operated under helium atmosphere at 1 atm. The ambient pressure operation facilitates applications to fluid samples. Three kinds of injectors are employed to feed randomly oriented crystals in aqueous solution or highly viscous fluid. Experiments on lysozyme crystals were performed by using the 10 keV XFEL of the SPring-8 Angstrom Compact free-electron LAser (SACLA). The structure of model protein lysozyme from 1 µm crystals at a resolution of 2.4 Å was obtained.

Time-resolved serial femtosecond crystallography using an X-ray free electron laser (XFEL) in conjunction with a photosensitive caged-compound offers a crystallographic method to track enzymatic reactions. Here we demonstrate the application of this method using fungal NO reductase, a heme-containing enzyme, at room temperature. Twenty milliseconds after caged-NO photolysis, we identify a NO-bound form of the enzyme, which is an initial intermediate with a slightly bent Fe-N-O coordination geometry at a resolution of 2.1 Å. The NO geometry is compatible with those analyzed by XFEL-based cryo-crystallography and QM/MM calculations, indicating that we obtain an intact Fe3+-NO coordination structure that is free of X-ray radiation damage. The slightly bent NO geometry is appropriate to prevent immediate NO dissociation and thus accept H− from NADH. The combination of using XFEL and a caged-compound is a powerful tool for determining functional enzyme structures during catalytic reactions at the atomic level., Using photosensitive caged-compound for femtosecond crystallography at X-ray free electron lasers would allow the structure determination of reaction intermediates. Here the authors demonstrate the feasibility of this approach with a caged NO-compound and present the initial NO-bound intermediate structure of cytochrome P450 nitric oxide reductase.

The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in "wires" inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inwardopen state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inwardopen state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.

Serial femtosecond crystallography (SFX) allows structures of proteins to be determined at room temperature with minimal radiation damage. A highly viscous matrix acts as a crystal carrier for serial sample loading at a low flow rate that enables the determination of the structure, while requiring consumption of less than 1 mg of the sample. However, a reliable and versatile carrier matrix for a wide variety of protein samples is still elusive. Here we introduce a hydroxyethyl cellulose-matrix carrier, to determine the structure of three proteins. The de novo structure determination of proteinase K from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of the praseodymium atom was demonstrated using 3, 000 diffraction images., 低ノイズ・低粘着性・低コストのタンパク質結晶輸送媒体を発見--生理条件に近いタンパク質の高分解能構造解析に期待--. 京都大学プレスリリース. 2017-04-20.

Snapshots of bacteriorhodopsin Bacteriorhodopsin is a membrane protein that harvests the energy content from light to transport protons out of the cell against a transmembrane potential. Nango et al. used timeresolved serial femtosecond crystallography at an x-ray free electron laser to provide 13 structural snapshots of the conformational changes that occur in the nanoseconds to milliseconds after photoactivation. These changes begin at the active site, propagate toward the extracellular side of the protein, and mediate internal protonation exchanges that achieve proton transport. Science , this issue p. 1552

Recently, significant technological innovations have enabled the measurement of both X-ray and electron diffraction from protein microcrystals. These new microcrystallography experiments are useful when large crystals cannot be obtained, but also in other cases, such as when large crystals suffer from long-range disorder, or when uniform perturbations need to be applied rapidly to the entire crystal volume. Optimizing the preparation of protein microcrystals for this new class of experiments presents new challenges for crystallographers, who have traditionally sought to grow large, single crystals. To better understand these new challenges, we optimized the production of microcrystalline samples of cyclophilin A (CypA), starting from conditions that produced millimeter scale crystals. Next, we used these microcrystals to determine CypA structures by serial femtosecond crystallography (SFX) at two XFEL lightsources, and by microcrystal electron diffraction (microED) in an electron cryomicroscope. Here, I will present our optimization strategy for protein microcrystallization, and compare the results of X-ray and electron microcrystallography experiments with CypA. I will focus on the unique caveats of sample delivery for each method, and compare the resulting structures. The goal will be to provide insight into which microcrystallography experiment is most appropriate for which types of samples, and to share our experience with sample preparation and delivery for each type of experiment.

Chemoenzymatic synthesis of peptides is a green and clean chemical reaction that offers high yields without using organic synthesis and serves as an alternative to traditional peptide synthesis methods. This report describes the chemoenzymatic synthesis of oligo(L-phenylalanine) mediated by proteinase K from Tritirachium album, which is one of the most widely used proteases in molecular biological studies. The synthesized linear oligo-phenylalanine showed a unique self-assembly in aqueous solutions. To further functionalize linear oligo(L-phenylalanine) as a low-molecular-weight gelator, it was cosynthesized with tris(2-aminoethyl)amine to obtain star-oligo(L-phenylalanine), which was bioconjugated to demonstrate its self-assembly into fluorescent fibers. The self-assembled fibers of star-oligo(L-phenylalanine) formed fibrous networks with various branching ratios, which depended on the molecular weights and molecular aspect ratios of star-oligo(L-phenylalanine). This is the first study to demonstrate that proteinase K is a suitable enzyme for chemoenzymatic cosynthesis of oligopeptides and star-shaped heteropeptides.

The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-Å resolution., 結晶を損傷しない新しいタンパク質結晶の輸送媒体を発見－タンパク質の結晶構造解析で新薬創生に一歩近づく. 京都大学プレスリリース. 2016-04-22.

Copper nitrite reductase (CuNiR) is involved in denitrification of the nitrogen cycle. Synchrotron X-rays rapidly reduce copper sites and decompose the substrate complex structure, which has made crystallographic studies of CuNiR difficult. Using femtosecond X-ray free electron lasers, we determined intact structures of CuNiR with and without nitrite. Based on the obtained structures, we proposed a redox-coupled proton switch model, which provides an explanation for proton-coupled electron transfer (PCET) in CuNiR. PCET is widely distributed through biogenic processes including respiratory and photosynthetic systems and is highly expected to be incorporated into bioinspired molecular devices. Our study also establishes the foundation for future studies on PCET in other systems.

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme–substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-A resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-A resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.

The CryoFibre, a crystal mounting tool, has been developed for protein cryocrystallography. The technique attaches single crystals to the tips of polyester fibres, allowing removal of excess liquid around each crystal. Single-wavelength anomalous dispersion phasing using a Cu Kα X-ray source (Cu SAD) was applied to crystals from five proteins without any derivatization, demonstrating a clear improvement in the success rate of Cu SAD compared with the conventional loop technique. In addition, a xylanase crystal on the surface of a synthetic zeolite as a hetero-epitaxic nucleant was directly mounted on the CryoFibre without separation treatment of the crystal from the zeolite. The crystal had a lower mosaicity than that observed using the conventional technique, indicating that the fibre technique is suitable for high-quality data collection from zeolite-mediated crystals.

The applicability of the heteroepitaxic nucleant method using molecular sieves (MS) to sparse matrix screening of protein crystallization was examined using xylanase as a test protein. The sparse matrix screening with MS provided three crystal forms I−III under 80% of conditions, which confirmed a much higher yield of crystal formation when compared to the conventional screening without MS. In the form I crystals, an MS-dependent improvement in X-ray diffraction quality was observed. Furthermore, form III of xylanase crystals was obtained only in the presence of MS 5A or 13X, demonstrating an MS-dependent packing-space expansion of protein crystals.

Ligand soaking of protein crystals is important for the preparation of heavy-atom derivative crystals for experimental phasing as well as for large-scale ligand screening in pharmaceutical developments. To facilitate laborious large-scale ligand screening, to reduce the risk of human contact with hazardous ligand reagents and to increase the success rate of the soaking experiments, a protein crystallization robot `Autolabo' has been developed and implemented in the high-throughput crystallization-to-structure pipeline at RIKEN SPring-8 Center. The main functions of this robotic system are the production of protein crystals for experiments, the ligand soaking of these crystals and the observation of soaked crystals. The separate eight-channel dispensers of Autolabo eliminate the cross-contamination of reagents which should be strictly avoided in the ligand-soaking experiment. Furthermore, the automated approach reduces physical damage to crystals during experiments when compared with the conventional manual approach, and thereby has the potential to yield better quality diffraction data. Autolabo's performance as a ligand-soaking system was evaluated with a crystallization experiment on ten proteins from different sources and a heavy-atom derivatization experiment on three proteins using a versatile cryoprotectant containing heavy-atom reagents as ligands. The crystallization test confirmed reliable crystal reproduction in a single condition and the capability for crystallization with nucleants to improve crystal quality. Finally, Autolabo reproducibly derivatized the test protein crystals with sufficient diffraction quality for experimental phasing and model building, indicating a high potentiality of this automated approach in ligand-soaking experiments.

HATODAS II is the second version of HATODAS (the Heavy-Atom Database System), which suggests potential heavy-atom reagents for the derivatization of protein crystals. The present expanded database contains 3103 heavy-atom binding sites, which is four times more than the previous version. HATODAS II has three new criteria to evaluate the feasibility of the search results: (1) potentiality scoring for the predicted heavy-atom reagents, (2) exclusion of the disordered amino acid residues based on the secondary structure prediction and (3) consideration of the solvent accessibility of amino acid residues from a homology model. In the point mutation option, HATODAS II suggests possible mutation sites into reactive amino acid residues such as Met, Cys and His, on the basis of multiple sequence alignments of homologous proteins. These new features allow the user to make a well informed decision as to the possible heavy-atom derivatization experiments of protein crystals.