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Although StayGold is a bright and highly photostable fluorescent protein (FP), its obligate dimer formation may prevent its application in molecular fusion and membrane targeting. With the objective of attaining monovalent as well as bright and photostable labeling, we engineered tandem dimers of StayGold to be dispersible. On the basis of the crystal structure of this FP, we disrupted the dimer interface to develop monomeric variants of StayGold. We applied the new StayGold tools to live cell imaging experiments using spinning-disk laser scanning confocal microscopy or structured illumination microscopy. We achieved cell-wide, high–spatiotemporal-resolution, and sustained imaging of subcellular dynamic events, including the targeting of endogenous condensin I to mitotic chromosomes at the onset of mitosis, the movement of the Golgi apparatus and its membranous derivatives along microtubule networks, the distribution of cortical filamentous actin near the plasma membrane, and the remolding of cristae membranes within mobile mitochondria.

Although numerous surgical techniques are employed to treat acute Stanford type A aortic dissection (ATAAD), controversy remains over which is the best procedure for aortic root reconstruction. Among the various techniques utilized, neomedia repair is considered to be more promising than adhesive-only repair for the treatment of a dissected aortic root. We experienced a series of neomedia sinus Valsalva repair using woven polyester fabric, and evaluated the aortic root diameter by computed tomography and severity of aortic valve insufficiency by transthoracic echocardiography. The aortic root diameter was well preserved with no progress of aortic valve insufficiency in the long-term period. Furthermore, we found that the fabric looked functioning new media in the findings obtained from the pathological examination of a neomedia repaired aortic wall sample that was obtained by chance from a patient during valve replacement surgery performed 10 years after aortic reconstruction for ATAAD. Neomedia repair using woven polyester fabric for ATAAD might facilitate the long-term durability of the surgically treated aortic root. (This is a translation of J Jpn Coll Angiol 2019; 59: 37-43.).

A rapid and reliable sample changer, SPACE-II, has been developed at the SPring-8 macromolecular crystallography beamline BL41XU. It enables samples to be exchanged in 16 s, of which its action accounts for only 11 s. Two years of operating SPACE-II demonstrated that the average number of sample exchanges per day was increased by 40% compared with the previous model, and it had an error rate of only 0.089%., Reducing the sample-exchange time is a crucial issue in maximizing the throughput of macromolecular crystallography (MX) beamlines because the diffraction data collection itself is completed within a minute in the era of pixel-array detectors. To this end, an upgraded sample changer, SPACE-II, has been developed on the basis of the previous model, SPACE (SPring-8 Precise Automatic Cryo-sample Exchanger), at the BL41XU beamline at SPring-8. SPACE-II achieves one sample-exchange step within 16 s, of which its action accounts for only 11 s, because of three features: (i) the implementation of twin arms that enable samples to be exchanged in one cycle of mount-arm action, (ii) the implementation of long-stroke mount arms that allow samples to be exchanged without withdrawal of the detector and (iii) the use of a fast-moving translation and rotation stage for the mount arms. By pre-holding the next sample prior to the sample-exchange sequence, the time was further decreased to 11 s in the case of automatic data collection, of which the action of SPACE-II accounted for 8 s. Moreover, the sample capacity was expanded from four to eight Uni-Pucks. The performance of SPACE-II has been demonstrated in over two years of operation at BL41XU; the average number of samples mounted on the diffractometer in one day was increased from 132 to 185, with an error rate of 0.089%, which counted incidents in which users could not continue with an experiment without recovery work by entering the experimental hutch. On the basis of these results, SPACE-II has been installed at three other MX beamlines at SPring-8 as of July 2019. The fast and highly reliable SPACE-II is now one of the most important pieces of infrastructure for the MX beamlines at SPring-8, providing users with the opportunity to fully make use of limited beamtime with brilliant X-rays.

In situ diffraction data collection using crystallization plates has been utilized for macromolecules to evaluate crystal quality without requiring additional sample treatment such as cryocooling. Although it is difficult to collect complete data sets using this technique due to the mechanical limitation of crystal rotation, recent advances in methods for data collection from multiple crystals have overcome this issue. At SPring-8, an in situ diffraction measurement system was constructed consisting of a goniometer for a plate, an articulated robot and plate storage. Using this system, complete data sets were obtained utilizing the small-wedge measurement method. Combining this system with an acoustic liquid handler to prepare protein–ligand complex crystals by applying fragment compounds to trypsin crystals for in situ soaking, binding was confirmed for seven out of eight compounds. These results show that the system functioned properly to collect complete data for structural analysis and to expand the capability for ligand screening in combination with a liquid dispenser.

Many cadherin family proteins are associated with diseases such as cancer. Since cell adhesion requires homodimerization of cadherin molecules, a small-molecule regulator of dimerization would have therapeutic potential. Herein, we describe identification of a P-cadherin-specific chemical fragment that inhibits P-cadherin-mediated cell adhesion. Although the identified molecule is a fragment compound, it binds to a cavity of P-cadherin that has not previously been targeted, indirectly prevents formation of hydrogen bonds necessary for formation of an intermediate called the X dimer and thus modulates the process of X dimerization. Our findings will impact on a strategy for regulation of protein-protein interactions and stepwise assembly of protein complexes using small molecules., Senoo et al. describe a chemical fragment that disrupts dimerization of P-cadherin. In doing so, this fragment inhibits cadherin-mediated cell-adhesion and aggregation.

The concept of minimally invasive cardiac surgery has been gradually adopted world-wide since its inception more than 2 decades ago. Recently, catheter intervention has been used in the treatment of structural heart disease. Most notably, minimally invasive transcatheter aortic valve implantation is now an established treatment option for aortic valve stenosis. There are three major approaches for minimally invasive aortic valve surgery: via median sternotomy, via the parasternal approach, and via the thoracotomy approach. All these approaches allow for a small skin incision and/or avoid full sternotomy. Moreover, several advanced variations with additional aortic procedures or totally endoscopic management have been developed. When considering each approach, low invasiveness must be balanced with safety, as surgeons broaden their insight of advanced medicine. Physical invasiveness is largely related to the surgical approach in minimally invasive surgery. We review the history and evolution of the different surgical approaches for minimally invasive aortic valve replacement.

The effect of utilizing high-energy X-rays for macromolecular crystallography of a radiation-sensitive metal enzyme was investigated. The resultant structure was evaluated in combination with visible absorption spectroscopic data. No explicit energy dependence of radiation damage was found in the range from 10 to 30 keV., To investigate the effect of high-energy X-rays on site-specific radiation-damage, low-dose diffraction data were collected from radiation-sensitive crystals of the metal enzyme cytochrome c oxidase. Data were collected at the Structural Biology I beamline (BL41XU) at SPring-8, using 30 keV X-rays and a highly sensitive pixel array detector equipped with a cadmium telluride sensor. The experimental setup of continuous sample translation using multiple crystals allowed the average diffraction weighted dose per data set to be reduced to 58 kGy, and the resulting data revealed a ligand structure featuring an identical bond length to that in the damage-free structure determined using an X-ray free-electron laser. However, precise analysis of the residual density around the ligand structure refined with the synchrotron data showed the possibility of a small level of specific damage, which might have resulted from the accumulated dose of 58 kGy per data set. Further investigation of the photon-energy dependence of specific damage, as assessed by variations in UV-vis absorption spectra, was conducted using an on-line spectrometer at various energies ranging from 10 to 30 keV. No evidence was found for specific radiation damage being energy dependent.

Background Sinus of Valsalva aneurysm (SVA) is relatively rare and commonly reported as a congenital anomaly. It is usually found in a single Valsalva sinus protruding into another cardiac chamber and is termed as intracardiac SVA. The aneurysm usually originates from the Valsalva sinus itself, and an orifice of the aneurysm is observed surrounded by the aortic wall. Thus, extracardiac multiple SVAs originating from the orifices with partial aortic wall defects are extremely rare. We report a very rare case of unruptured extracardiac SVAs in both left and right coronary sinuses originating from the aortic annulus. Case presentation A 76-year-old Japanese male was diagnosed with enlarged Valsalva sinuses by transthoracic echocardiography during follow-up for peripheral artery disease. Five years after careful observation, gradual SVA enlargement and moderate aortic insufficiency were observed. He underwent modified Bentall’s procedure, with an uneventful postoperative course. Intraoperatively, SVAs were found in the left lateral half of the left and right coronary sinuses of Valsalva on both sides of the commissure between the left and right coronary cusps. Aortic walls were missing at the SVA floor adjacent to the aortic annulus. Pathological examination revealed only mild atherosclerotic changes of the aortic wall near the SVAs. The cause was estimated as either focal degeneration of the sinuses of Valsalva just above the aortic annulus or congenital anomaly, or combination of both of them. Conclusions We report on the case of unruptured extracardiac multiple SVAs missing aortic orifice just above the annulus. No similar case presentation was found in the literature. In this paper, we present details of operative findings and procedures, which will aid in procedure selection.

Damage-free structures of the resting state of one of the most studied copper nitrite reductases were obtained independently by X-ray free-electron laser (XFEL) and neutron crystallography, representing the first direct comparison of neutron and XFEL structural data for any protein. In addition, damage-free structures of the reduced and nitrite-bound forms have been obtained to high resolution from cryogenically maintained crystals by XFEL crystallography., Copper-containing nitrite reductases (CuNiRs) that convert NO2 − to NO via a CuCAT–His–Cys–CuET proton-coupled redox system are of central importance in nitrogen-based energy metabolism. These metalloenzymes, like all redox enzymes, are very susceptible to radiation damage from the intense synchrotron-radiation X-rays that are used to obtain structures at high resolution. Understanding the chemistry that underpins the enzyme mechanisms in these systems requires resolutions of better than 2 Å. Here, for the first time, the damage-free structure of the resting state of one of the most studied CuNiRs was obtained by combining X-ray free-electron laser (XFEL) and neutron crystallography. This represents the first direct comparison of neutron and XFEL structural data for any protein. In addition, damage-free structures of the reduced and nitrite-bound forms have been obtained to high resolution from cryogenically maintained crystals by XFEL crystallography. It is demonstrated that AspCAT and HisCAT are deprotonated in the resting state of CuNiRs at pH values close to the optimum for activity. A bridging neutral water (D2O) is positioned with one deuteron directed towards AspCAT Oδ1 and one towards HisCAT N∊2. The catalytic T2Cu-ligated water (W1) can clearly be modelled as a neutral D2O molecule as opposed to D3O+ or OD−, which have previously been suggested as possible alternatives. The bridging water restricts the movement of the unprotonated AspCAT and is too distant to form a hydrogen bond to the O atom of the bound nitrite that interacts with AspCAT. Upon the binding of NO2 − a proton is transferred from the bridging water to the Oδ2 atom of AspCAT, prompting electron transfer from T1Cu to T2Cu and reducing the catalytic redox centre. This triggers the transfer of a proton from AspCAT to the bound nitrite, enabling the reaction to proceed.

An automated data-collection system named ZOO has been developed. This system enabled faster data collection, facilitated advanced data-collection and data-processing techniques, and permitted the collection of higher quality data., Owing to the development of brilliant microfocus beamlines, rapid-readout detectors and sample changers, protein microcrystallography is rapidly becoming a popular technique for accessing structural information from complex biological samples. However, the method is time-consuming and labor-intensive and requires technical expertise to obtain high-resolution protein crystal structures. At SPring-8, an automated data-collection system named ZOO has been developed. This system enables faster data collection, facilitates advanced data-collection and data-processing techniques, and permits the collection of higher quality data. In this paper, the key features of the functionality put in place on the SPring-8 microbeam beamline BL32XU are described and the major advantages of this system are outlined. The ZOO system will be a major driving force in the evolution of the macromolecular crystallography beamlines at SPring-8.

Nitric oxide (NO) reductase from the fungus Fusarium oxysporum is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N(2)O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate (I), a key state to promote N–N bond formation and N–O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of I. TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe–NO coordination in I, with an elongated Fe–NO bond length (Fe–NO = 1.91 Å, Fe–N–O = 138°) in the absence of NAD(+). TR-infrared (IR) spectroscopy detects the formation of I with an N–O stretching frequency of 1,290 cm(−1) upon hydride transfer from NADH to the Fe(3+)–NO enzyme via the dissociation of NAD(+) from a transient state, with an N–O stretching of 1,330 cm(−1) and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of I is characterized by a singly protonated Fe(3+)–NHO(•−) radical. The current findings provide conclusive evidence for the N(2)O generation mechanism via a radical–radical coupling of the heme nitroxyl complex with the second NO molecule.

Valve sparing aortic root replacement( VSRR) is currently an established option for patients with annuloaortic ectasia( AAE). A newly designed Valsalva graft, the J Graft Shield Neo Valsalva, was used for VSRR in three cases. All operations were successful and postoperative courses were uneventful. Graft designs closer in shape to the native Valsalva may contribute to the improvement of late outcomes in VSRR.

Summary Bile acids are metabolites of cholesterol that facilitate lipid digestion and absorption in the small bowel. Bile acids work as agonists of receptors to regulate their own metabolism. Bile acids also regulate other biological systems such as sugar metabolism, intestinal multidrug resistance, and adaptive immunity. However, numerous physiological roles of bile acids remain undetermined. In this study, we solved the crystal structure of human serine hydroxymethyltransferase (hSHMT) in complex with an endogenous secondary bile acid glycine conjugate. The specific interaction between hSHMT and the ligand was demonstrated using mutational analyses, biophysical measurements, and structure-activity relationship studies, suggesting that secondary bile acid conjugates may act as modulators of SHMT activity., Graphical abstract, Highlights • The crystal structures of hSHMT in complex with secondary bile acid glycine conjugate • Specific interactions between hSHMT and secondary bile acid conjugate were validated • Biological role of bile acids as modulators for one-carbon metabolism is suggested, Molecular Interaction; Structural Biology; Metabolic Engineering

Highly accurate SR structures and damage-free XFEL structures provide detailed insight into catalytic mechanism of CuNiR., Copper-containing nitrite reductases (CuNiRs), encoded by nirK gene, are found in all kingdoms of life with only 5% of CuNiR denitrifiers having two or more copies of nirK. Recently, we have identified two copies of nirK genes in several α-proteobacteria of the order Rhizobiales including Bradyrhizobium sp. ORS 375, encoding a four-domain heme-CuNiR and the usual two-domain CuNiR (Br2DNiR). Compared with two of the best-studied two-domain CuNiRs represented by the blue (AxNiR) and green (AcNiR) subclasses, Br2DNiR, a blue CuNiR, shows a substantially lower catalytic efficiency despite a sequence identity of ~70%. Advanced synchrotron radiation and x-ray free-electron laser are used to obtain the most accurate (atomic resolution with unrestrained SHELX refinement) and damage-free (free from radiation-induced chemistry) structures, in as-isolated, substrate-bound, and product-bound states. This combination has shed light on the protonation states of essential catalytic residues, additional reaction intermediates, and how catalytic efficiency is modulated.

Cryocrystallography, which is commonly used in macromolecular crystallography, may sometimes reduce the quality of diffraction data and the visibility of crystals owing to frost adhesion. A device has been developed to remove frost by drizzling liquid nitrogen over the crystals, which enabled noise reduction of diffraction images and the centering of crystals with low visibility owing to frost adhesion., Cryocrystallography is a technique that is used more often than room-temperature data collection in macromolecular crystallography. One of its advantages is the significant reduction in radiation damage, which is especially useful in synchrotron experiments. Another advantage is that cryopreservation provides simple storage of crystals and easy transportation to a synchrotron. However, this technique sometimes results in the undesirable adhesion of frost to mounted crystals. The frost produces noisy diffraction images and reduces the optical visibility of crystals, which is crucial for aligning the crystal position with the incident X-ray position. To resolve these issues, a computer-controlled device has been developed that drizzles liquid nitrogen over a crystal to remove frost. It was confirmed that the device works properly, reduces noise from ice rings in diffraction images and enables the centering of crystals with low visibility owing to frost adhesion.

Many cadherin family proteins are associated with diseases such as cancer. Since cell adhesion requires homodimerization of cadherin molecules, a small-molecule regulator of dimerization would have therapeutic potential. Herein, we describe identification of a P-cadherin-specific chemical fragment that inhibits P-cadherin-mediated cell adhesion. Although the identified molecule is a fragment compound, it binds to a cavity of P-cadherin that has not previously been targeted, indirectly prevents formation of hydrogen bonds necessary for formation of an intermediate called the X dimer and thus modulates the on-rate of X dimerization. Our findings will impact on a strategy for kinetic regulation of protein-protein interactions and stepwise assembly of protein complexes using small molecules.

Serial crystallography, in which single-shot diffraction images are collected, has great potential for protein microcrystallography. Although serial femtosecond crystallography (SFX) has been successfully demonstrated, limited beam time prevents its routine use. Inspired by SFX, serial synchrotron crystallography (SSX) has been investigated at synchrotron macromolecular crystallography beamlines. Unlike SFX, the longer exposure time of milliseconds to seconds commonly used in SSX causes radiation damage. However, in SSX, crystals can be rotated during the exposure, which can achieve efficient coverage of the reciprocal space. In this study, mercury single-wavelength anomalous diffraction (Hg-SAD) phasing of the luciferin regenerating enzyme (LRE) was performed using serial synchrotron rotation crystallography. The advantages of rotation and influence of dose on the data collected were evaluated. The results showed that sample rotation was effective for accurate data collection, and the optimum helical rotation step depended on multiple factors such as multiplicity and partiality of reflections, exposure time per rotation angle and the contribution from background scattering. For the LRE microcrystals, 0.25° was the best rotation step for the achievable resolution limit, whereas a rotation step larger than or equal to 1° was favorable for Hg-SAD phasing. Although an accumulated dose beyond 1.1 MGy caused specific damage at the Hg site, increases in resolution and anomalous signal were observed up to 3.4 MGy because of a higher signal-to-noise ratio., 微小タンパク質結晶からの効率的な構造解析法 : 凍結した試料を回転させる「SS-ROX法」を確立、汎用化へ. 京都大学プレスリリース. 2017-01-12.

Foreign metallic bodies in the mediastinum are relatively rare and may cause life-threatening damages to multiple organs in the mediastinum; therefore, the foreign body warrants urgent or elective removal. When the foreign body remains in the posterior mediastinum surrounded by important organs, for example, the heart or the great vessels, it is critical to determine the right approach for its safe removal. Here, we present the successful removal of a sewing needle from the posterior mediastinum using multidetector computed tomography that helped identify the foreign body’s location and its relationship with the surrounding organs.

The bending magnet beamline BL38B1 at SPring-8 is equipped with X-ray optics consisting of the SPring-8 standard double crystal monochromator (DCM) with Si (111) and a vertically bent cylindrical mirror, which produce available energy ranging from 6 to 17 keV and photon flux of 8.6 × 1010 Ph/sec at 12.4 keV. These moderate beam properties are suited to high throughput cryogenic macromolecular crystallography (MX) using moderately sized crystals (over 100 µm) and non-cryo-cooled MX methodology that takes advantage of the lower X-ray dose rate. However, along with the recent research trends the target crystal sizes have tended to become smaller and smaller, and are currently less than 100 µm. To resolve this issue, we achieved an upgrade of beamline optics for higher flux and higher density X-rays and development of apparatuses for precise diffraction data collection.

We report a case of an 84-year-old Japanese female patient with transcatheter valve embolization treated with thoracic endovascular stenting. She was diagnosed with severe aortic stenosis and referred to our hospital. Transapical transcatheter aortic valve implantation (TA-TAVI) was selected because she had multiple comorbidities and peripheral vascular disease. During TA-TAVI, the transcatheter valve (TV) came loose and lodged in the distal aortic arch. The TV was floating and unstable, thereby affecting her hemodynamics. After a second TV was placed in the appropriate position with no trouble, we tried fixing the TV using a thoracic endovascular stent-graft. The procedure was successful, and the patient recovered well. The cause of embolization seemed to be associated with a relatively mild calcification of the native aortic valve. Although this complication is rare, several recovery procedures should be prepared.

A nearly on-axis UV–visible absorption spectrometer was developed at SPring-8 that enables spectroscopic analysis of the X-ray-exposed volume of a crystal during X-ray diffraction data collection., UV–visible absorption spectroscopy is useful for probing the electronic and structural changes of protein active sites, and thus the on-line combination of X-ray diffraction and spectroscopic analysis is increasingly being applied. Herein, a novel absorption spectrometer was developed at SPring-8 BL26B2 with a nearly on-axis geometry between the X-ray and optical axes. A small prism mirror was placed near the X-ray beamstop to pass the light only 2° off the X-ray beam, enabling spectroscopic analysis of the X-ray-exposed volume of a crystal during X-ray diffraction data collection. The spectrometer was applied to NO reductase, a heme enzyme that catalyzes NO reduction to N2O. Radiation damage to the heme was monitored in real time during X-ray irradiation by evaluating the absorption spectral changes. Moreover, NO binding to the heme was probed via caged NO photolysis with UV light, demonstrating the extended capability of the spectrometer for intermediate analysis.

Purpose Mortality in patients with ruptured abdominal aortic aneurysms (rAAAs) has remained high despite advances in interventions. Endovascular aneurysm repair (EVAR) was recently developed for treatment of rAAAs. In this study, we assessed our endovascular strategy including a double-balloon technique for rAAA. Methods We analyzed 12 consecutive patients with rAAAs who were treated by our double-balloon technique and endovascular strategy from March 2013 to July 2016. Results The 30-day and 1-year mortality rates were both 17%. The mean times from admission to arrival at the hybrid operating room, from admission to aortic occlusion, and from admission to completion of EVAR were 46.8, 63.5, and 110.0 minutes, respectively. Conclusion This study indicates that the herein-described double-balloon endovascular technique is feasible for use in the management of rAAA.

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 observation of O2 in a time-frozen structure using serial femtosecond rotation crystallography of the as-isolated oxidized enzyme provides long-awaited clear-cut evidence for the mode of O2 binding in CuNiRs. This provides an insight into how CuNiR from A. xylosoxidans can function as an oxidase, reducing O2 to H2O2, or as a superoxide dismutase (SOD) since it was shown to have ∼56% of the dismutase activity of the bovine SOD enzyme some two decades ago., Synchrotron-based X-ray structural studies of ligand-bound enzymes are powerful tools to further our understanding of reaction mechanisms. For redox enzymes, it is necessary to study both the oxidized and reduced active sites to fully elucidate the reaction, an objective that is complicated by potential X-ray photoreduction. In the presence of the substrate, this can be exploited to construct a structural movie of the events associated with catalysis. Using the newly developed approach of serial femtosecond rotation crystallography (SF-ROX), an X-ray damage-free structure of the as-isolated copper nitrite reductase (CuNiR) was visualized. The sub-10 fs X-ray pulse length from the SACLA X-ray free-electron laser allowed diffraction data to be collected to 1.6 Å resolution in a ‘time-frozen’ state. The extremely short duration of the X-ray pulses ensures the capture of data prior to the onset of radiation-induced changes, including radiolysis. Unexpectedly, an O2 ligand was identified bound to the T2Cu in a brand-new binding mode for a diatomic ligand in CuNiRs. The observation of O2 in a time-frozen structure of the as-isolated oxidized enzyme provides long-awaited clear-cut evidence for the mode of O2 binding in CuNiRs. This provides an insight into how CuNiR from Alcaligenes xylosoxidans can function as an oxidase, reducing O2 to H2O2, or as a superoxide dismutase (SOD) since it was shown to have ∼56% of the dismutase activity of the bovine SOD enzyme some two decades ago.

Photosynthesis converts light energy into biologically useful chemical energy vital to life on Earth. The initial reaction of photosynthesis takes place in photosystem II (PSII), a 700-kilodalton homodimeric membrane protein complex which catalyses photo-oxidation of water into dioxygen through an S-state cycle of the oxygen evolving complex (OEC). The structure of PSII has been solved by X-ray diffraction (XRD) at 1.9-ångström (Å) resolution, which revealed that the OEC is a Mn4CaO5-cluster coordinated by a well-defined protein environment1. However, extended X-ray absorption fine structure (EXAFS) studies showed that the manganese cations in the OEC are easily reduced by X-ray irradiation2, and slight differences were found in the Mn–Mn distances between the results of XRD1, EXAFS3–7 and theoretical studies8–14. Here we report a ‘radiation-damage-free’ structure of PSII from Thermosynechococcus vulcanus in the S1 state at a resolution of 1.95 Å using femtosecond X-ray pulses of the SPring-8 ångström compact free-electron laser (SACLA) and a huge number of large, highly isomorphous PSII crystals. Compared with the structure from XRD, the OEC in the X-ray free electron laser structure has Mn–Mn distances that are shorter by 0.1–0.2 Å. The valences of each manganese atom were tentatively assigned as Mn1D(III), Mn2C(IV), Mn3B(IV) and Mn4A(III), based on the average Mn–ligand distances and analysis of the Jahn–Teller axis on Mn(III). One of the oxo-bridged oxygens, O5, has significantly longer Mn–O distances in contrast to the other oxo-oxygen atoms, suggesting that it is a hydroxide ion instead of a normal oxygen dianion and therefore may serve as one of the substrate oxygen atoms. These findings provide a structural basis for the mechanism of oxygen evolution, and we expect that this structure will provide a blueprint for design of artificial catalysts for water oxidation.

OBJECTIVES: The optimal strategy for coronary revascularization in patients with renal dysfunction remains undefined. As coronary artery bypass grafting (CABG) may be associated with higher mortality, less invasive percutaneous coronary intervention (PCI) may be preferred. To date, only limited information has been published regarding the effects of severe renal dysfunction on the outcome after CABG and PCI. To address this limitation, we analysed the clinical outcomes after CABG or PCI in patients with severe renal dysfunction not on chronic haemodialysis (HD). METHODS: Among patients enrolled in the CREDO-Kyoto (Coronary Revascularization Demonstrating Outcome Study in Kyoto) Registry (a multicentre PCI/CABG registry in Japan), we identified 374 patients with multivessel disease and an estimated glomerular filtration rate

BACKGROUND Few data exist on the results of transcatheter aortic valve implantation (TAVI) via the transfemoral approach in small slightly built Japanese patients with severe aortic stenosis who are ineligible or at high-risk for conventional surgical aortic valve replacement (SAVR). Therefore, the purpose was to investigate the early outcomes of TAVI using the transiliofemoral approach in Japan. METHODS AND RESULTS Between June 2010 and June 2013, 21 consecutive patients (mean age, 81.0 years; 81.0% female) underwent TAVI with Edwards SAPIEN XT valves using the transiliofemoral approach. The mean body surface area was 1.44±0.15m(2). The device success rate was 90.5%. Although 2 patients did not meet the echocardiographic criteria for device success, no failure to deliver and deploy a valve occurred. The mean effective aortic valve area increased from 0.54±0.12cm(2) at baseline to 1.46±0.29cm(2) after the procedure (P

A Si(111) winged crystal has been designed for sagittal focusing of synchrotron radiation X-rays. The results of performance tests at beamlines are reported., A Si(111) winged crystal has been designed to minimize anticlastic bending and improve sagittal focusing efficiency. The crystal was thin with wide stiffening wings. The length-to-width ratio of the crystal was optimized by finite element analysis, and the optimal value was larger than the ‘golden value’. The analysis showed that the slope error owing to anticlastic bending is less than the Darwin width. The X-rays were focused two-dimensionally using the crystal and a tangentially bent mirror. The observed profiles of the focal spot agreed well with the results of a ray-tracing calculation in the energy range from 8 to 17.5 keV. X-ray diffraction measurements with a high signal-to-noise ratio using this focusing system were demonstrated for a small protein crystal.

Beamline BL44XU at SPring-8 is operated by the Institute for Protein Research of Osaka University. The beamline is designed for X-ray crystallography of large biological macromolecular assemblies. Here we show its detailed performances, results, and the ongoing upgrade plans.

At SPring-8 MX beamlines, a remote access system has been developed and started user operation in 2010. The system has been developed based on an automated data collection and data management architecture utilized for the confirmed scheme of SPring-8 mail-in data collection. Currently, further improvement to the remote access and automation which covers data processing and analysis are being developed.

Protein crystallography beamlines at the two major Japanese synchrotron facilities (SPring-8 and Photon Factory) employ different crystal-exchange robots. The robots SPACE and PAM, installed at SPring-8 and the Photon Factory, respectively, accelerate crystal exchange and assist in the efficient use of beamtime. SPACE also supports customizable automatic data collection, in which an X-ray beam is irradiated at a manually determined position during the crystal evaluation process. However, this data collection requires special crystal-mounting screw pins made of polyacetal, which are not compatible with PAM. In order to give users the flexibility to collect diffraction data sets at either of the two synchrotrons, we have developed a compatible cassette system. Pins stored in the cassette can be mounted or demounted using either SPACE or PAM, and are compatible with the customizable automatic data collection provided by SPACE. Users can handle the pins and cassettes easily, employing common tools and techniques for efficient protein cryocrystallography experiments.

SPACE (SPring-8 precise automatic cryo-sample exchanger) is an automated sample exchanger for cryo-cooled protein crystals, developed at SPring-8. Since the start of its operation, SPACE has been continuously improved and upgraded to cope with the requirements of new beamlines and users. One important upgrade of SPACE provides support for conventional metal-base pins, which are attached to the goniometer head magnetically. Other hardware improvements include increasing the sample storage capacity. The upgraded version of SPACE, as reported here, is continuously operated and is an essential component of beamline operation at SPring-8.

The region spanning residues 95-146 of the rotavirus nonstructural protein NSP4 from the asymptomatic human strain ST3 has been purified and crystallized and diffraction data have been collected to a resolution of 2.6 angstrom. Several attempts to solve the structure by the molecular-replacement method using the available tetrameric structures of this domain were unsuccessful despite a sequence identity of 73% to the already known structures. A more systematic approach with a dimer as the search model led to an unexpected pentameric structure using the program Phaser. The various steps involved in arriving at this molecular-replacement solution, which unravelled a case of subtle variation between different oligomeric states unknown at the time of solving the structure, are presented in this paper.

Leukotriene (LT) C(4) and its metabolites, LTD(4) and LTE(4), are involved in the pathobiology of bronchial asthma. LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA(4) binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA(4).

By combining the use of relatively large crystals and an X-ray free-electron laser, a radiation damage–free three-dimensional structure of a radiation-sensitive protein (bovine cytochrome oxidase) was solved at 1.9-A resolution. We report a method of femtosecond crystallography for solving radiation damage–free crystal structures of large proteins at sub-angstrom spatial resolution, using a large single crystal and the femtosecond pulses of an X-ray free-electron laser (XFEL). We demonstrated the performance of the method by determining a 1.9-A radiation damage–free structure of bovine cytochrome c oxidase, a large (420-kDa), highly radiation-sensitive membrane protein.