X-ray absorption near edge structures of DNA or its components around the oxygen K-shell edge

The initial process of radiation damage in DNA was investigated by measuring the X-ray absorption near edge structures (XANES) within the energy region around the oxygen K-shell absorption edge for DNA, cytosine and 2-deoxy-d-ribose. Irradiation and XANES experiments were performed with the BL23SU soft X-ray beamline, using synchrotron radiation from the 8 GeV electron storage ring at SPring-8. Samples were mounted on gold-coated plates in a vacuum chamber. The XANES spectra were obtained by measuring the photoelectron current of the samples. 2-Deoxy-d-ribose was exposed to X rays at the absorption peak corresponding to the oxygen (O) 1s-->sigma* transition energy (538 eV); the XANES spectra were obtained after each irradiation. DNA and cytosine, possessing characteristic XANES spectra, both had two major energy bands corresponding to the O 1s-->pi* and 1s-->sigma* transitions. Two new peaks appeared and gradually increased in the XANES spectra of 2-deoxy-d-ribose during irradiation. These results suggest that C-O bonds in 2-deoxy-d-ribose are transformed to C=O bonds by O 1s-->sigma* transition, suggesting that the molecules undergo chemical changes into carbonyl-containing compounds.     

Serial femtosecond crystallography at the SACLA: breakthrough to dynamic structural biology

X-ray crystallography visualizes the world at the atomic level. It has been used as the most powerful technique for observing the three-dimensional structures of biological macromolecules and has pioneered structural biology. To determine a crystal structure with high resolution, it was traditionally required to prepare large crystals (> 200 μm). Later, synchrotron radiation facilities, such as SPring-8, that produce powerful X-rays were built. They enabled users to obtain good quality X-ray diffraction images even with smaller crystals (ca. 200–50 μm). In recent years, one of the most important technological innovations in structural biology has been the development of X-ray free electron lasers (XFELs). The SPring-8 Angstrom Compact free electron LAser (SACLA) in Japan generates the XFEL beam by accelerating electrons to relativistic speeds and directing them through in-vacuum, short-period undulators. Since user operation started in 2012, we have been involved in the development of serial femtosecond crystallography (SFX) measurement systems using XFEL at the SACLA. The SACLA generates X-rays a billion times brighter than SPring-8. The extremely bright XFEL pulses enable data collection with microcrystals (ca. 50–1 μm). Although many molecular analysis techniques exist, SFX is the only technique that can visualize radiation-damage-free structures of biological macromolecules at room temperature in atomic resolution and fast time resolution. Here, we review the achievements of the SACLA-SFX Project in the past 5 years. In particular, we focus on: (1) the measurement system for SFX; (2) experimental phasing by SFX; (3) enzyme chemistry based on damage-free room-temperature structures; and (4) molecular movie taken by time-resolved SFX.     

In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology?

The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography.     

Canadian macromolecular crystallography facility: a suite of fully automated beamlines

The Canadian light source is a 2.9 GeV national synchrotron radiation facility located on the University of Saskatchewan campus in Saskatoon. The small-gap in-vacuum undulator illuminated beamline, 08ID-1, together with the bending magnet beamline, 08B1-1, constitute the Canadian Macromolecular Crystallography Facility (CMCF). The CMCF provides service to more than 50 Principal Investigators in Canada and the United States. Up to 25% of the beam time is devoted to commercial users and the general user program is guaranteed up to 55% of the useful beam time through a peer-review process. CMCF staff provides "Mail-In" crystallography service to users with the highest scored proposals. Both beamlines are equipped with very robust end-stations including on-axis visualization systems, Rayonix 300 CCD series detectors and Stanford-type robotic sample auto-mounters. MxDC, an in-house developed beamline control system, is integrated with a data processing module, AutoProcess, allowing full automation of data collection and data processing with minimal human intervention. Sample management and remote monitoring of experiments is enabled through interaction with a Laboratory Information Management System developed at the facility.     

Crystal structure of pullulanase: evidence for parallel binding of oligosaccharides in the active site

The crystal structures of Klebsiella pneumoniae pullulanase and its complex with glucose (G1), maltose (G2), isomaltose (isoG2), maltotriose (G3), or maltotetraose (G4), have been refined at around 1.7-1.9A resolution by using a synchrotron radiation source at SPring-8. The refined models contained 920-1052 amino acid residues, 942-1212 water molecules, four or five calcium ions, and the bound sugar moieties. The enzyme is composed of five domains (N1, N2, N3, A, and C). The N1 domain was clearly visible only in the structure of the complex with G3 or G4. The N1 and N2 domains are characteristic of pullulanase, while the N3, A, and C domains have weak similarity with those of Pseudomonas isoamylase. The N1 domain was found to be a new type of carbohydrate-binding domain with one calcium site (CBM41). One G1 bound at subsite -2, while two G2 bound at -1 approximately -2 and +2 approximately +1, two G3, -1 approximately -3 and +2 approximately 0', and two G4, -1 approximately -4 and +2 approximately -1'. The two bound G3 and G4 molecules in the active cleft are almost parallel and interact with each other. The subsites -1 approximately -4 and +1 approximately +2, including catalytic residues Glu706 and Asp677, are conserved between pullulanase and alpha-amylase, indicating that pullulanase strongly recognizes branched point and branched sugar residues, while subsites 0' and -1', which recognize the non-reducing end of main-chain alpha-1,4 glucan, are specific to pullulanase and isoamylase. The comparison suggested that the conformational difference around the active cleft, together with the domain organization, determines the different substrate specificities between pullulanase and isoamylase.     

The beamlines of ELETTRA and their application to structural biology

Protein crystallographers are nowadays regular users of synchrotron radiation (SR) facilities for several applications. The goal of majority of users is simply to extract more accurate, higher resolution data from existing crystals; they use monochromatic radiation and the rotation method, in order to get a complete survey of the reciprocal space in a short time. In fact the brilliance of SR is essential, due to the weak scattering power of the samples, and because of their sensibility to radiation damage. Over the last few years, however, a general increase of interest for measurements at multiple wavelengths, which exploit the anomalous dispersion for the phase problem (multiwavelength anomalous diffraction — MAD), has generated the need of intense tuneable sources. For these applications, the emphasis is on accurate measurements of the small differences between the intensities of Bragg reflections at various energies across the absorption edge of an element present in the sample. The macromolecular diffraction beamline at ELETTRA, which is now running routinely since spring 1995, has been designed to provide a high flux — highly collimated tuneable X-rays source in the spectral range between 4 and 25 keV. The radiation source is the 57-pole wiggler, which delivers a very intense radiation up to 25 keV, and is shared and used simultaneously with the small angle X-ray scattering (SAXS) beamline. The front-end filter system has a cut-off energy at about 4 keV. The beamline optics consists of a pseudo-channel-cut double-crystal monochromator followed by a double focusing toroidal mirror. The tunability and the stability of the monochromator allows the user to perform MAD experiments, and for this purpose, a fluorescence probe for the exact calibration of the absorption edge is available on-line. The experimental station is based on an imaging plate area detector from MarResearch, with a sensible area of 345 mm in diameter. A cooled N₂-stream is available to cool the sample crystal in order to reduce the radiation damage. SAXS is an experimental technique used to derive structural information about supra-molecular assemblies, amorphous materials and partly ordered systems (e.g. size and shape of large molecules). The high-flux SAXS beamline at ELETTRA is mainly intended for time-resolved studies on fast structural transitions in the sub-millisecond time region in solutions and in partly ordered systems, triggered by external or process parameters, with a SAXS resolution between 10 and 1400 Å in real space. The source is the already mentioned 57-pole and the SAXS beamline accepts three discrete energies of its spectrum, namely 5.4, 8 and 16 keV. The beamline optics consists of a flat double-crystal monochromator and a double focusing toroidal mirror. A multi-purpose sample stage, movable along an optical table in order to optimise the sample to detect distance, allows to perform fast time-resolved relaxation studies based on temperature- or pressure-jumps as well as stopped flow experiments. Moreover, the users have option to install their own specialised sample surrounding equipment. The optimisation of the beamline with respect to high-flux and consequently high-flux density, allows to perform the following experiments: low contrast solution scattering, grazing incidence surface diffraction, micro-spot scanning, X-ray fluorescence analysis, time-resolved studies 11 s, simultaneous small- and wide-angle measurements on gels, liquid crystals, biopolymers, amorphous materials, muscles.This revised version was published online in October 2005 with corrections to the Cover Date.     

Structural changes of actin-bound myosin heads after a quick length change in frog skeletal muscle

Changes in the x-ray diffraction pattern from a frog skeletal muscle were recorded after a quick release or stretch, which was completed within one millisecond, at a time resolution of 0.53 ms using the high-flux beamline at the SPring-8 third-generation synchrotron radiation facility. Reversibility of the effects of the length changes was checked by quickly restoring the muscle length. Intensities of seven reflections were measured. A large, instantaneous intensity drop of a layer line at an axial spacing of 1/10.3 nm(-1) after a quick release and stretch, and its partial recovery by reversal of the length change, indicate a conformational change of myosin heads that are attached to actin. Intensity changes on the 14.5-nm myosin layer line suggest that the attached heads alter their radial mass distribution upon filament sliding. Intensity changes of the myosin reflections at 1/21.5 and 1/7.2 nm(-1) are not readily explained by a simple axial swing of cross-bridges. Intensity changes of the actin-based layer lines at 1/36 and 1/5.9 nm(-1) are not explained by it either, suggesting a structural change in actin molecules.     

Crystallization and preliminary X-ray diffraction studies of the human adenovirus serotype 2 proteinase with peptide cofactor.

Recombinant human adenovirus serotype 2 proteinase (both native and selenomethionine-substituted) has been crystallized in the presence of the serotype 12, 11-residue peptide cofactor. The crystals (space group P3(1)21 or P3(2)21, one molecule per asymmetric unit, a = b = 41.3 angstrum, c = 197.0 angstrum) grew in solutions containing 20-40% 2-methyl-2,4-pentanediol (MPD), 0.1-0.2 M sodium citrate, and 0.1 M sodium HEPES, pH 5.0-7.5. Diffraction data (84% complete to 2.2 angstrum resolution with Rmerge of 0.0335) have been measured from cryopreserved native enzyme crystals with the Argonne blue (1,024 x 1,024 pixel array) charge-coupled device detector at beamline X8C at the National Synchrotron Light Source (operated by Argonne National Laboratory's Structural Biology Center). Additionally, diffraction data from selenomethionine-substituted proteinase, 65% complete to 2.0 angstrum resolution with Rmerge values ranging 0.05-0.07, have been collected at three X-ray energies at and near the selenium absorption edge. We have determined three of the six selenium sites and are initiating a structure solution by the method of multiwavelength anomalous diffraction phasing.     

Automation of macromolecular crystallography beamlines

The production of three-dimensional crystallographic structural information of macromolecules can now be thought of as a pipeline which is being streamlined at every stage from protein cloning, expression and purification, through crystallisation to data collection and structure solution. Synchrotron X-ray beamlines are a key section of this pipeline as it is at these that the X-ray diffraction data that ultimately leads to the elucidation of macromolecular structures are collected. The burgeoning number of macromolecular crystallography (MX) beamlines available worldwide may be enhanced significantly with the automation of both their operation and of the experiments carried out on them. This paper reviews the current situation and provides a glimpse of how a MX beamline may look in the not too distant future.     

Curative effects of combination therapy with lentinan and interleukin-2 against established murine tumors,and the role of CD8-positive T cells

The antitumor activity of a combination of an antitumor polysaccharide, lentinan (a 1–3 glucan with 1–6 branches), and interleukin-2 (IL-2) was evaluated against established MBL-2 lymphoma and S908.D2 sarcoma at i.d. sites. Treatment of the MBL-2-tumor-bearing BDF1 mice with lentinan and IL-2 induced complete regression of tumor in 87.5% of mice treated. In contrast, treatments using either lentinan or IL-2 alone failed to induce complete regression of tumor, although temporal growth inhibition of tumor was observed about in half of the mice treated. Improvements of antitumor effects by the combination of lentinan and IL-2 were also observed in the MBL-2/B6 and S908.D2/B10.D2 systems. Expression of the antitumor effects of lentinan/IL-2 treatments required the intact T cell compartment, because the effects were not observed when nude mice were used. In the MBL-2/B6 system, the antitumor action of lentinan/IL-2 treatment was abolished in mice treated with antibody to CD8 antigen, whereas antibodies to CD4 or NK1.1 were ineffective. Furthermore, augmented tumor-specific cytotoxic T lymphocyte (CTL) activity was observed in regional lymph node cells of the mice after lentinan and IL-2 administration. These data indicate that the antitumor effects of lentinan/IL-2 are mediated by CD8⁺ CTL but not by CD4⁺ T cells or NK1.1⁺ NK/LAK cells, and suggest that this combined therapy may be effective against even established tumors that are resistant to IL-2 therapy.Abbreviations B6 C57BL/6 - BDF1 C57BL/6 × DBA/2 F1 - Lyt2 murine CD8, Lyt2.1. allele of murine CD8 - Lyt2.2 allele of murine CD8 - Lyt3 murine CD8 - L3T4 murine CD4     

High performance liquid chromatographic quantitation of carboxypeptidase activity secreted by human hep G2 cells

A new high performance liquid chromatographic method has been developed for the determination of carboxypeptidase N activity which quantitates the furylacryloyl-alanine released by enzymatic cleavage of furylacryloyl-alanyl-lysine or furylacryloyl-alanyl-arginine. A short isocratic gradient elutes the substrate and product in less than 7 min and multiple analyses are facilitated by an automatic sample injector. The microassay readily detects and quantitates carboxypeptidase N activity secreted into culture medium. It was determined that approximately 1 X 10(6) Hep G2 cells at early confluence secreted 1 ng of carboxypeptidase N in 24 h. The microassay will also detect as little as 51 pg of purified carboxypeptidase N or 8 pg of carboxypeptidase B.     

Functional characterization of neuropeptide 26RFa receptors GPR103A and GPR103B in zebrafish,Danio rerio

The hypothalamic neuropeptide 26RFa is the most recently identified member of the RFamide peptide family, and this 26RFa signaling system has been shown to be implicated in regulating a variety of physiological processes. In zebrafish，26RFa and two putative receptors, DrGPR103A and DrGPR103B, have been in silico identified, and in vivo data derived from overexpression and loss of function mutation experiments suggest the 26RFa signaling system plays an important role in the hypothalamic regulation of sleep. However, the biochemical and pharmacological information on DrGPR103A/B receptors is still unknown. Here, after cloning of cDNAs of two putative 26RFa receptor genes, DrGPR103A and B, from the total RNA of zebrafish whole body, functional assays demonstrated that both receptors were activated by synthetic zebrafish 26RFa neuropeptide, leading to a significant increase in CRE-driven luciferase activity and intracellular Ca²⁺ mobilization in a G_αq inhibitor- and G_αi/o inhibitor-sensitive manner. Upon activation by 26RFa, DrGPR103A and B evoked ERK1/2 phosphorylation and underwent internalization. Further functional determination also revealed that zebrafish kisspeptin-1 exhibited a slight potency for activating both DrGPR103A and B, and vice versa, zebrafish 26RFa also showed some activity at zebrafish GPR54A and B. Our findings provided evidence that zebrafish GPR103A and B are two functional G_αq- and G_αi/o-dually coupled receptors for 26RFa, enabling the further elucidation of the endocrinological roles of zebrafish 26RFa signaling system in the regulation of physiological activities.     

Recombination between kappa chain genetic markers and the Lyt-3 locus

Recombination has been detected for the first time between chromosome 6 loci controlling kappa chain expression in normal mouse serum immunoglobulin and the Lyt-3 locus. The recombination event occurred at the 26th or 27th backcross generation during the derivation of the Lyt-2a, Lyt-3a-congenic line B6.PL(85NS). The line is now homozygous for the Lyt-2a, Lyt-3a allele(s) at N30F13 and homozygous animals express the Igk-Ef1b allele derived from C57BL/6. The frequency of recombination has been estimated to be 0.30% based on the present results and previous studies in which no recombination was detected. The results rule out the hypothesis that the Lyt-3 locus itself controls the light chain phenotype observed in normal serum immunoglobulin.     

Influence of Burkitt's lymphoma and primary B cells on latent gene expression by the nonimmortalizing P3J-HR-1 strain of Epstein-Barr virus.

The Epstein-Barr virus (EBV) genes expressed in B lymphocytes immortalized in vitro or in Burkitt's lymphoma (BL) cells infected in vivo have been characterized previously; however, the viral products which are essential for immortalization or for establishment of EBV latency are still not known. To approach this question, we compared the kinetics of expression of EBV nuclear antigens and the two EBV-encoded small RNAs, EBER1 and EBER2, after infection of primary B cells or EBV genome-negative BL cells with either an immortalizing EBV strain (B95-8) or the nonimmortalizing deletion mutant (HR-1). Following infection of primary cells with B95-8 virus, EBV nuclear antigen (EBNA)-2 was expressed first, followed by EBNA-1, -3, and -4 (also called leader protein [LP]) and the two small RNAs. Infection of EBV genome-negative BL cells with the same strain of virus resulted in a similar pattern of gene expression, except that the EBNAs appeared together and more rapidly. EBERs were not apparent in one BL cell line converted by B95-8. The only products detected after infection of primary B lymphocytes with the HR-1 deletion mutant were the EBNA-4 (LP) family and trace amounts of EBER1. Although HR-1 could express neither EBNA-1, EBNA-3, nor EBER2 in primary cells, all these products were expressed rapidly after HR-1 infection of EBV genome-negative BL cell lines. The results indicate that the mutation in HR-1 virus affects immortalization not only through failure to express EBNA-2, a gene which is deleted, but also indirectly by curtailing expression of several other EBV genes whose coding regions are intact in the HR-1 virus and normally expressed during latency. The pattern of latent EBV gene expression after HR-1 infection is dependent on the host cell, perhaps through products specific for the cell cycle or the state of B-cell differentiation.     

Crystallographic analysis of AcrB

A His-tagged derivative of the multidrug efflux pump AcrB could be crystallized in three different space groups (R3, R32 and P321). Experimental MAD-phasing maps from R32 AcrB(His) crystals were obtained to a resolution of 3.5 A. Datasets of native and substrate soaked AcrB(His) crystals were collected at the Swiss Light Source X06SA beamline up to a resolution of 2.7 A and refinement of these data provided good quality electron density maps, which allowed us to complement the published AcrB structure (PDB code 1iwg). Introduction of amino acids 860-865 and 868 lacking in the 1iwg structure and deletion of a highly disordered region (amino acids 669-678) improved R(free) and average B factors in the 2.7 A model. We could not identify significant densities indicating specific antibiotic binding sites in the AcrB R32 space group datasets under the soaking conditions tested.     

High-throughput crystallization-to-structure pipeline at RIKEN SPring-8 Center

A high-throughput crystallization-to-structure pipeline for structural genomics was recently developed at the Advanced Protein Crystallography Research Group of the RIKEN SPring-8 Center in Japan. The structure determination pipeline includes three newly developed technologies for automating X-ray protein crystallography: the automated crystallization and observation robot system "TERA", the SPring-8 Precise Automatic Cryosample Exchanger "SPACE" for automated data collection, and the Package of Expert Researcher's Operation Network "PERON" for automated crystallographic computation from phasing to model checking. During the 5 years following April, 2002, this pipeline was used by seven researchers to determine 138 independent crystal structures (resulting from 437 purified proteins, 234 cryoloop-mountable crystals, and 175 diffraction data sets). The protocols used in the high-throughput pipeline are described in this paper.     

Synthesis,crystallization and preliminary crystallographic analysis of a 52-nucleotide DNA/2′-OMe-RNA oligomer mimicking 10–23 DNAzyme in the complex with a substrate

A 52-nucleotide DNA/2′-OMe-RNA oligomer mimicking 10–23 DNAzyme in the complex with its substrate was synthesized, purified and crystallized by the hanging-drop method using 0.8 M sodium potassium tartrate as a precipitant. A data set to 1.21 Å resolution was collected from a monocrystal at 100 K using synchrotron radiation on a beamline BL14.1 at BESSY. The crystal belonged to the P2₁ group with unit-cell a = 49.42, b = 24.69, c = 50.23, β = 118.48.