Christoph Hermes

Upgrade of the small-angle X-ray scattering beamline X33 at the European Molecular Biology Laboratory, Hamburg

The small-angle X-ray scattering beamline X33 of the European Molecular Biology Laboratory (EMBL) at the DORIS III storage ring [Deutsches Elektronen Synchrotron (DESY) Hamburg] was used for more than two decades to study the structure of non-crystalline biological systems. During recent years the beamlines scope has changed and is now predominantly used to analyze solutions of biological macromolecules. Owing to renewed interest in solution scattering studies from the biological community, the workload on the beamline has steadily increased. A major upgrade of X33 was performed to improve the beamline stability and data quality, to shorten the measurement time and to ensure user-friendly operation. The upgrade involved all major components of the beamline, including the optical system (monochromator, mirror, slits, beam monitors), electronics, control and acquisition software, X-ray detector system and the sample environment. The upgrade improved the brilliance by a factor of about three and the measuring time was reduced by a factor of seven. The knowledge and experience gained during the implementation of the upgrades to X33, may aid the design process for the BioSAXS beamline to be constructed for the PETRA-3 facility at DESY.

Evidence for discrete Cd(SCys)4 units in cadmium phytochelatin complexes from EXAFS spectroscopy

The principal structural features of multinuclear complexes of cadmium(II) with the plant-peptide phytochelatin have been deduced from Cd-EXAFS (EXAFS = extended X-ray absorption fine structure) data: (i) Cd(SCys)4 coordination, (ii) in contrast to Cd-metallothioneins, no formation of cadmium ‘clusters’ and (iii) non-coordinating carboxylate groups.

Instrumental setup for high-throughput small- and wide-angle solution scattering at the X33 beamline of EMBL Hamburg

A setup is presented for automated high-throughput measurements of small-angle X-ray scattering (SAXS) from macromolecular solutions on the bending-magnet beamline X33 of EMBL at the storage ring DORIS-III (DESY, Hamburg). A new multi-cell compartment allows for rapid switching between in-vacuum and in-air operation, for digital camera assisted control of cell filling and for colour sample illumination. The beamline is equipped with a Pilatus 1 M-W pixel detector for SAXS and a Pilatus 300 k-W for wide-angle scattering (WAXS), and results from the use of the Pilatus detectors for scattering studies are reported. The setup provides a broad resolution range from 100 to 0.36 nm without the necessity of changing the sample-to-detector distance. A new optimized robotic sample changer is installed, permitting rapid and reliable automated sample loading and cell cleaning with a required sample volume of 40 µl. All the devices are fully integrated into the beamline control software system, ensuring fully automated and user-friendly operation (attended, unattended and remote) with a throughput of up to 15 measurements per hour.

Automation of the EMBL Hamburg protein crystallography beamline BW7B

The EMBL Hamburg Outstation currently operates five synchrotron beamlines for protein crystallography. The strongest of these beamlines is the fixed-energy beamline BW7B which receives about half of the radiation (1.5 mrad) from a 56 pole wiggler located at the DORIS III storage ring at the German synchrotron facility DESY. Over the last years this beamline has been upgraded and equipped with a fully automated crystallographic end-station and a robotic sample changer. The current set-up allows for remote operation, controlled from the users area, of sample mounting, centering and data collection of pre-frozen crystals mounted in Hampton-type cryovials on magnetic caps. New software and intuitive graphical user interfaces have been developed that control the complete beamline set-up. Furthermore, algorithms for automatic sample centering based on UV fluorescence are being developed and combined with strategy programs in order to further automate the collection of entire diffraction data sets.

X-RAY INSTRUMENTATION FOR A FOCUSED WIGGLER BEAMLINE AT THE EMBL OUTSTATION HAMBURG

A flexible setup consisting of two independent water‐cooled X‐ray monochromators providing radiation for two separate beamlines (A and B) has been installed on the multipole wiggler BW7 at the DORIS III storage ring. The first optical element of both lines is a Rh‐coated SiC mirror that reduces the heat load on subsequent energy defining elements. Line B, which will be predominantly used for protein crystallography, is optimized for flux and employs a triangular monochromator and segmented glass mirror for horizontal and vertical focusing, respectively; it can be operated in a wavelength range from 0.5 to 1.8 A. Line A features a high‐energy resolution fixed exit, nondispersive double monochromator, optimized for accurate and fast energy scans. It will mainly be used for anomalous scattering, EXAFS, and small‐angle scattering. The use of encoders, intelligent stepper/dc‐motor controllers, and position sensitive beam monitors allow a comfortable and fast alignment of all axes. Both experimental hutches are...

Overview of the tunable beamlines for protein crystallography at the EMBL Hamburg Outstation; an analysis of current and future usage and developments.

The EMBL Hamburg Outstation currently operates two tunable protein crystallography beamlines suitable for single and multiple anomalous diffraction (SAD/MAD) experiments. The first beamline, designated X31, is located on a bending magnet of the DORIS III storage ring whereas the second beamline, BW7A, is positioned at a multipole wiggler at the same storage ring. X31 is equipped with an energy stabilization device to ensure constant wavelength during longer data-collection periods. The in-house built crystallographic end-station is now equipped with a Mar345 imaging-plate scanner as a detector. The wiggler beamline BW7A features a novel sagitally focusing monochromator. The end-station used here has also been developed and built in-house. The beamline is currently operated with a Mar 165 CCD detector. In this paper the hardware and software developments of the last years will be summarized and the outlook for substantial upgrades will be given. The future plans include the design and construction of a third tunable beamline, designated X12, for protein crystallography. The development of automated beamlines for protein crystallography is of particular importance with respect to structural genomics initiatives. The analysis of the projects of the last years shows the wide range of anomalous scatterer used on the tunable beamlines thus demonstrating the need of a wide range of accessible energies and fast and reliable energy changes.

A small and robust active beamstop for scattering experiments on high-brilliance undulator beamlines

Using an indirect detection scheme, a small-size beamstop was developed to accurately measure X-ray beam flux in a wide energy range with reduced radiation level on the electronics and significantly increased in-beam lifetime.

Cobalt X-ray absorption spectroscopy of glucose isomerase

Introduction Glucose isomerases catalyze the isomerization of D-glucose to D-fructose and of D-xylose to D-xylulose. They are of considerable interest in the industrial production of high-fructose corn syrup. Glucose isomerase from Streptomyces albus (GI) is a tetramer (Mr = 172.000) with four identical subunits [1,2]. Each subunit contains two distinct metal bindin of Co2+, Mn f sites, a tight binding site (B-site) and a weaker binding site (A-site). Addition + or Mg2’ * increases the catalytic activity as well as the stability of the enzyme.

New Beamlines For Protein Crystallography At The EMBL‐Outstation Hamburg

Three new beamlines for Protein Crystallography were built up on a bending magnet fan of the DORIS III storage ring. A 10 mrad wide fan of white Synchrotron Radiation (SR) is evenly distributed among 3 individual stations: X12, a central, wavelength‐tunable station intended for anomalous scattering experiments (MAD) and fixed wavelength, high intensity stations symmetrically on either side. The fixed wavelength beamlines X11 & X13 comprise triangular, asymmetrically cut Si (111) monochromators as horizontally focusing optical elements. The tunable station is based on a fixed‐exit, horizontally focusing double crystal monochromator system. Vertical focusing is achieved on all three lines by trapezoidal shaped, continuous Rh‐coated mirrors which can be dynamically bent. In all three lines the X‐ray beam can be examined at various points on its way through the optical system by removable screens and PIN‐diode based intensity monitors. Purpose built crystallographic end‐stations complete the set‐up. The desig...

A Structural Model for the Soybean Lipoxygenase-1 Iron Site

In this communication the striking similarity between the EXAFS of soybean lipoxygenase-1 and that of a methanol-solution of a well-characterized iron-polyimidazole model compound is reported. This similarity is a strong corroboration of the structure proposed earlier for the non-heme iron active site in lipoxygenase (S. Navaratnam, M.C. Feiters, M. Al-Hakim, J.C. Allen, G.A. Veldink, J.F.G. Vliegenthart, Biochim. Biophys. Acta, 1988, 956, 70)