Michel Fodje

Calcium Stiffens Archaeal Rad51 Recombinase from Methanococcus voltae for Homologous Recombination.

Archaeal RadA or Rad51 recombinases are close homologues of eukaryal Rad51 and DMC1. These and bacterial RecA orthologues play a key role in DNA repair by forming helical nucleoprotein filaments in which a hallmark strand exchange reaction between homologous DNA substrates occurs. Recent studies have discovered the stimulatory role by calcium on human and yeast recombinases. Here we report that the strand exchange activity but not the ATPase activity of an archaeal RadA/Rad51 recombinase from Methanococcus voltae (MvRadA) is also subject to calcium stimulation. Crystallized MvRadA filaments in the presence of CaCl2 resemble that of the recently reported ATPase active form in the presence of an activating dose of KCl. At the ATPase center, one Ca2+ ion takes the place of two K+ ions in the K+-bound form. The terminal phosphate of the nonhydrolyzable ATP analogue is in a staggered conformation in the Ca2+-bound form. In comparison, an eclipsed conformation was seen in the K+-bound form. Despite the changes in the ATPase center, both forms harbor largely ordered L2 regions in essentially identical conformations. These data suggest a unified stimulation mechanism by potassium and calcium because of the existence of a conserved ATPase center promiscuous in binding cations.

08B1-1: an automated beamline for macromolecular crystallography experiments at the Canadian Light Source

Beamline 08B1-1 is a recently commissioned bending-magnet beamline at the Canadian Light Source. The beamline is designed for automation and remote access. Together with the undulator-based beamline 08ID-1, they constitute the Canadian Macromolecular Crystallography Facility. This paper describes the design, specifications, hardware and software of beamline 08B1-1. A few scientific results using data obtained at the beamline will be highlighted.

MxDC and MxLIVE: software for data acquisition, information management and remote access to macromolecular crystallography beamlines.

An integrated computer software system for on-site and remote collection of macromolecular crystallography (MX) data at the Canadian Light Source (CLS) is described. The system consists of an integrated graphical user interface for data collection and beamline control [MX Data Collector (MxDC)] which provides experiment-focused control of beamline devices, and a laboratory information management system [MX Laboratory Information Virtual Environment (MxLIVE)] for managing sample and experiment information through a web browser. The system allows remote planning and transmission of sample and experiment parameters to the beamline through MxLIVE, on-site or remote data collection through MxDC guided by information from MxLIVE, and remote monitoring and download of experimental results through MxLIVE. The system is deployed and in use on both MX beamlines at the CLS which constitute the Canadian Macromolecular Crystallography Facility.

Cyclo-linopeptide K butanol disolvate monohydrate.

The title compound, C56H83N9O11S·2C4H10O·H2O, is a butanol–water solvate of the cyclolinopeptide cyclo(Metsulfone1-Leu2–Ile3–Pro4–Pro5–Phe6–Phe7–Val8–Ile9) (henceforth referred to as CLP-K) which was isolated from flax oil. All the amino acid residues are in an l configuration based on the CORN rule. The cyclic nonapeptide exhibits eight trans peptide bonds and one cis peptide bond observed between the two proline residues. The conformation is stabilized by an α- and a β-turn, each containing an N—H⋯O hydrogen bond between the carbonyl group O atom of the first residue and the amide group H atom of the fourth (α-turn) and the third residue (β-turn), repectively. In the crystal, the components of the structure are linked by intermolecular N—H⋯O and O—H⋯O hydrogen bonds into a two-dimensional network parallel to (001). The –C(H2)OH group of one of the butanol solvent molecules is disordered over two sets of sites with refined occupancies of 0.863 (4) and 0.137 (4).

VP8, the Major Tegument Protein of Bovine Herpesvirus 1, Interacts with Cellular STAT1 and Inhibits Interferon Beta Signaling

ABSTRACT The UL47 gene product, VP8, is the most abundant tegument protein of bovine herpesvirus 1 (BoHV-1). Previously, we demonstrated that a UL47-deleted BoHV-1 mutant (BoHV1-ΔUL47) exhibits 100-fold-reduced virulence in vitro and is avirulent in vivo. In this study, we demonstrated that VP8 expression or BoHV-1 infection inhibits interferon beta (IFN-β) signaling by using an IFN-α/β-responsive plasmid in a luciferase assay. As transducer and activator of transcription (STAT) is an essential component in the IFN-signaling pathways, the effect of VP8 on STAT was investigated. An interaction between VP8 and STAT1 was established by coimmunoprecipitation assays in both VP8-transfected and BoHV-1-infected cells. Two domains of VP8, amino acids 259 to 482 and 632 to 686, were found to be responsible for its interaction with STAT1. The expression of VP8 did not induce STAT1 ubiquitination or degradation. Moreover, VP8 did not reduce STAT1 tyrosine phosphorylation to downregulate IFN-β signaling. However, the expression of VP8 or a version of VP8 (amino acids 219 to 741) that contains the STAT1-interacting domains but not the nuclear localization signal prevented nuclear accumulation of STAT1. Inhibition of nuclear accumulation of STAT1 also occurred during BoHV-1 infection, while nuclear translocation of STAT1 was observed in BoHV1-ΔUL47-infected cells. During BoHV-1 infection, VP8 was detected in the cytoplasm at 2 h postinfection without any de novo protein synthesis, at which time STAT1 was already retained in the cytoplasm. These results suggest that viral VP8 downregulates IFN-β signaling early during infection, thus playing a role in overcoming the antiviral response of BoHV-1-infected cells. IMPORTANCE Since VP8 is the most abundant protein in BoHV-1 virions and thus may be released in large amounts into the host cell immediately upon infection, we proposed that it might have a function in the establishment of conditions suitable for viral replication. Indeed, while nonessential in vitro, it is critical for BoHV-1 replication in vivo. In this study, we determined that VP8 plays a role in downregulation of the antiviral host response by inhibiting IFN-β signaling. VP8 interacted with and prevented nuclear accumulation of STAT1 at 2 h postinfection in the absence of de novo viral protein synthesis. Two domains of VP8, amino acids 259 to 482 and 632 to 686, were found to be responsible for this interaction. These results provide a new functional role for VP8 in BoHV-1 infection and a potential explanation for the lack of viral replication of the UL47 deletion mutant in cattle.

Cyclo­linopeptide B methanol tris­olvate

The title compound, C56H83N9O9S·3CH3OH, is a methanol trisolvate of the cyclolinopeptide cyclo(Met1—Leu2—Ile3—Pro4—Pro5—Phe6—Phe7—Val8—Ile9) (henceforth referred to as CLP-B), which was isolated from flaxseed oil. All the amino acid residues are in an l-configuration based on the CORN rule. The cyclic nonapeptide exhibits eight trans peptide bonds and one cis peptide bond observed between the two proline residues. The conformation is stabilized by an α-turn and two consecutive β-turns each containing a N—H⋯O hydrogen bond between the carbonyl group O atom of the first residue and the amide group H atom of the fourth (α-turn) or the third residue (β-turns), repectively. In the crystal, the components of the structure are linked by N—H⋯O and O—H⋯O hydrogen bonds into chains parallel to the a axis.

Crystallization, Preliminary X-ray Diffraction and Structure Analysis of Thermotoga maritima Mannitol Dehydrogenase

Diffraction data have been collected from a crystal of Thermotoga maritima mannitol dehydrogenase at the Canadian Light Source. The crystal diffracted to 3.3 A resolution and belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 83.43, b = 120.61, c = 145.76 A. The structure is likely to be solved by molecular replacement.

Celebrating the 10th anniversary of CMCF operations

The Canadian Macromolecular Crystallography Facility (CMCF) supports more than 70 protein crystallographers from across Canada. It consists of an insertion device beamline (CMCF-ID) and a bending magnet beamline (CMCF-BM). The first diffraction image was acquired at the CMCF-ID beamline on March 18, 2006, and the first paper containing data collected at CMCF-ID was published in December 2006 [1]. CMCF-BM was commissioned in 2011, and is a general-purpose crystallography beamline having characteristics that support the protein and chemical crystallography communities. Both beamlines are fully automatized and operate mainly through remote access to the CMCF facility [2]. Up to 25% of available beamtime is reserved for commercial users. To date, more than 450 peer reviewed articles used data collected at the CMCF. Additionally, some 860 PDB deposits and 6 patents have originated from the CMCF. Since 2010, we have been running the Annual CLS Mx Data Collection School which has trained more than 140 participants (http://cmcf.lightsource.ca/school/). After a successful 10 years of operation, the CMCF-ID beamline will undergo an upgrade to establish micro-beam capabilities [3]. After completion, the focused beam at the sample will have a much smaller size of 50 × 5 μm^2 (H x V) with flux at 12 keV of ~10^13 [photons/s], allowing measurement of X-ray diffraction patterns from much smaller crystals than possible presently. The beamline will be equipped with a very fast sample changer and an ultra-low noise photon counting detector, allowing shutterless operation of the beamline. The first phase of the upgrade will include installation of a new PILATUS 3 6M detector that will become operational in May 2017, while an ISARA robot will be installed at the beamline in the autumn of 2017. For the second phase of the upgrade, a new longer in-vacuum undulator and new optics will be installed in approximately 3 years. To substantially increase the flux produced by the CMCF-BM beamline, we will additionally be upgrading its double crystal monochromator with multilayers.

Putative thioredoxin Trx1 from Thermosipho africanus strain TCF52B: expression, purification and structural determination using S-SAD.

Thioredoxin is a small ubiquitous protein that plays a role in many biological processes. A putative thioredoxin, Trx1, from Thermosipho africanus strain TCF52B, which has low sequence identity to its closest homologues, was successfully cloned, overexpressed and purified. The protein was crystallized using the microbatch-under-oil technique at 289 K in a variety of conditions; crystals grown in 0.2 M MgCl2, 0.1 M bis-tris pH 6.5, 25%(w/v) PEG 3350, which grew as irregular trapezoids to maximum dimensions of 1.2 × 1.5 × 0.80 mm, were used for sulfur single-wavelength anomalous dispersion analysis. The anomalous sulfur signal could be detected to 2.83 Å resolution using synchrotron radiation on the 08B1-1 beamline at the Canadian Light Source. The crystals belonged to space group P212121, with unit-cell parameters a = 40.6, b = 41.5, c = 56.4 Å, α = β = γ = 90.0°.

AutoProcess:Automated strategy calculation, data processing & structure solution

Two critical aspect of macromolecular crystallography experiments are (1) Determining the optimal parameters and strategy for collecting good quality data and (2) Optimal processing of the collected data to obtain to facilitate structure determination. These tasks can be daunting to inexperienced crystallographers and often lead to inefficiencies as valuable beam-time is used up. To support automation, remote access and high-throughput crystallography, we have developed a software system for automation of all data processing tasks required at the synchrotron. AutoProcess, is layered on the XDS data processing package and makes use of other utilities such as BEST from the European Molecular Biology Laboratory (EMBL), CCP4 utilities and SHELX. The software can be used from the command line as a standalone application but can also be run as a service on a high-performance computing cluster, and integrated into beamline control and information management systems such as MxDC and MxLIVE to allows users to determine the optimal strategy for data collection, and/or process full datasets with the click of a button. Users are presented with a graphical data processing report as well as reflection output files in popular formats automatically. For small molecule and peptide structures, an unrefined initial structure with an electron density map is automatically generated using only the raw diffraction images and the chemical composition of the molecule. Future developments will include sub-structure solution for MAD/SAD/SIRAS data. The software is freely available under an open-source license from the authors. The Canadian Light Source is supported by the Natural Sciences and Engineering Research Council of Canada, the National Research Council Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan.