No abstract

A high-throughput, general purpose robotic system has been developed which transports flash-cooled crystals securely and safely, automatically mounts crystals on most goniometers, optically centers them in the x-ray beam, screens for diffraction quality, collects and processes diffraction images, recovers the crystals and stores them in a liquid nitrogen dewar. The system is a refinement of the proven actor system from abbott laboratories [1]. Magnetic crystal storage and transport magazines are designed to hold up to 60 crystals in a standard dry shipper with easy access to any of the positions. The system is based on a general purpose 5-axis programmable robot that allows the system to be used in an infinite number of configurations including horizontal, vertical or virtually any other goniometer axis orientation. The unique non-magnetic end-effector securely grips standard hampton pins and places them on a ìglide goniometer magnet without the need to move cryosystream nozzles or beam stops. The ìglide goniometer translates the crystal ± 3 mm in all directions in as little as 2 micron increments for centering. All operations are controlled by director, a software tool which totally automates the high-throughput process of selection, centering, screening, collection and retrieval of crystals. The entire procedure has been optimized for high-throughput crystallography at synchrotron beamlines and in the home lab. References [1] Muchmore, S.W., Olson, J., Jones, R., Pan, J., Blum, M., Greer, J., Merrick, S.M., Magdalinos, P., & Nienaber, V. (2000) Structure 8, R243-R246. Bruker introduced lens-coupled CCD detectors two years ago. Since then we have reported on their use for macromolecular crystallography. However small molecule crystallographers have become interested in these devices, particularly because of their active area. At ALS, for example the large area (300 mm diameter) allows for positioning the detector at 2θ = 0 and collecting high resolution data most efficiently. This is despite the bending magnet source spectrum limitation of high flux at 14 KeV, or lower, i.e. longer wavelength than Mo Kα. Calibration of these detectors will be discussed and example of data collection will be given. The Canadian Light Source (CLS), a third-generation 2.9 GEV synchrotron light source, is currently under construction. The protein crystallography beamline is one of the seven beamlines to be constructed initially (of 35 beamlines at total capacity). The CMCF is planned to be a 50 m long undulator beamline to facilitate studying small crystals (approx. 50 mm) and crystals with large unit cells (approx. 1000 angstroms). Mad phasing will be one of the major applications of the beamline. Spectral range: 6.5 kev -18 kev (1.91 -0.69 angstroms), calculated flux at 12 kev (1.0 angstrom), approx. 1 x E14 ph/s/0.1/percent/bw and the focused spot size at the sample (horizontal x vertical) 0.15 mm x 0.05 mm (fwhm) generated from the 7th harmonic. Advanced user interface and control software, together with fully automated robotic...

obtained and 6 structures were determined. Among them, Bs139 protein functions as phosphoribosylglycinamide formyltransferase (GART), an important enzyme in the de novo pathway of purine biosynthesis. Bs139 crystal diffracted to 2.5 Å resolution at home Xray source and the structure was determined by molecular replacement (MR). Bs154 protein is a putative deoxyuridine 5'-triphosphate nucleotidehydrolase (dUTPase), which plays important role in DNA replication. Se-YosS crystal diffraction datasets were collected at Beijing Synchrotron Radiation Facility (BSRF) and the structure was determined by multi-wavelength anomalous diffraction (MAD) method.

anhydrous and hydrated models for the complex, the subunits, and the assembly from the constituents [4][5][6][7][8]. Moreover, the crystallographic availability of this MDa complex provides the opportunity to apply modern scattering and hydrodynamic modeling approaches to such huge entities, including assemblage of the complex from its constituents, and to check the extensive reduction steps to be adopted for modeling. We tackled the following problems: (i) comparison of the SAXS-or EM-based conventional or ab initio models for the HBL complex with up-to-date crystallographic data, (ii) modelling the HBL complex from constituents, (iii) realistic assumptions or predictions regarding the contribution of hydration, (iv) search for any discrepancies between solution and crystal data. For modelling, primarily the programs DAMMIN, HYDRO, HYDCRYST and several modifications of the approaches were applied, in addition to usage of templates and superimpositions; results were checked by prediction of structural and hydrodynamic data. The most serious problems arose from amino acids missing in the crystallographic data base. We eventually managed to explain the observed discrepancies by the residues absent in the crystal structure of the linker chains; these residues are obviously located in the central core of the complex. . et al., J. Biol. Chem. 1996, 271, 18695; Biopolymers, 1998, 45, 289; Biophys. J. 2004 Biophys. J. , 87, 1173 [5] Zipper P., Durchschlag H., J. Appl. Crystallogr. 2000, 33, 788; 2003, 36, 509; Physica A, 2002, 314, 613; J. Biol. Phys. 2007, 33, 523; Eur. Biophys. J. 2010, 39, 481. [6] Zipper P. et al., in: Analytical Ultracentrifugation (Scott D.J. et al., eds.), RSC, Cambridge, 2005, p. 320; Prog. Coll. Polym. Sci. 2002, 119, 141;2004, 127, 126; 2006, 131, 41 We have begun using a hybrid pixel detector (HPD), specifically the Dectris Pilatus 100K, in home lab single crystal X-ray diffraction experiments. In order to assess the utility of such a device for the home lab, we have studied the performance of this device for both small molecule and protein data collection experiments with copper radiation. We will present results comparing HPD data collection to conventional CCD data collection as well as results comparing conventional data collection to "shutterless" data collection in terms of data quality and increased throughput. Conjugative plasmid transfer is an important way for horizontal gene spread (e.g. antibiotic resistance genes) [1]. It can lead to the increase of bacteria with multiple antibiotic resistances. The plasmid conjugation process in Gramnegative bacteria has been studied in detail, whereas little information is available about the corresponding mechanisms in Gram-positive bacteria [2]. The transfer region of our Gram-positive multiple antibiotic resistance plasmid pIP501 is organized in an operon encoding fifteen putative transfer proteins. The transfer region of pIP501 encodes a putative simplified type IV secretion system (T4SS), as three pIP501-encoded Tra proteins show ...