Open access article in Journal of Synchrotron Radiation

Current events

2009-11-01

Advances and synergy of high-pressure sciences at synchrotron sources

Liu, H. — 2009-11-01

Introductory overview to the special issue papers on high-pressure sciences and synchrotron radiation.

Use of a hexapod in diffraction measurements of substrate-supported crystals of organic semiconductors

Yang, L. — 2009-11-01

Thin films of organic semiconductor prepared on substrates generally contain crystals that have one common crystal plane parallel to the substrate but random in-plane orientations. In diffraction measurements of these structures, it is often required to anchor the X-ray beam on a fixed spot on the sample, such as an optically visible crystallite or island. Here, a hexapod is used in place of a traditional multi-circle diffractometer to perform area-detector-based diffraction measurements on an actual device that contains 6,13-bis(triisopropylsilyethynyl)-pentacene (TIPS-pentacene) crystals. The hexapod allows for sample rotations about any user-defined rotation center. Two types of complex sample motions have been programmed to characterize the structure of the TIPS-pentacene crystal: an in-plane powder average has been performed at a fixed grazing-incident angle to determine the lattice parameters of the crystal; then the in-plane component of the scattering vector was continuously rotated in transmission geometry to determine the local crystal orientation.

A decade of user operation on the macromolecular crystallography MAD beamline ID14-4 at the ESRF

McCarthy, A.A. — 2009-11-01

ID14-4 at the ESRF is the first tunable undulator-based macromolecular crystallography beamline that can celebrate a decade of user service. During this time ID14-4 has not only been instrumental in the determination of the structures of biologically important molecules but has also contributed significantly to the development of various instruments, novel data collection schemes and pioneering radiation damage studies on biological samples. Here, the evolution of ID14-4 over the last decade is presented, and some of the major improvements that were carried out in order to maintain its status as one of the most productive macromolecular crystallography beamlines are highlighted. The experimental hutch has been upgraded to accommodate a high-precision diffractometer, a sample changer and a large CCD detector. More recently, the optical hutch has been refurbished in order to improve the X-ray beam quality on ID14-4 and to incorporate the most modern and robust optical elements used at other ESRF beamlines. These new optical elements will be described and their effect on beam stability discussed. These studies may be useful in the design, construction and maintenance of future X-ray beamlines for macromolecular crystallography and indeed other applications, such as those planned for the ESRF upgrade.

Current events

2009-09-01

Comments on Determination of X-ray flux using silicon pin diodes by R. L. Owen et al. (2009). J. Synchrotron Rad. 16, 143–151

Krumrey, M. — 2009-09-01

Response to Krumrey's Comments on Determination of X-ray flux using silicon pin diodes by R. L. Owen et al. (2009). J. Synchrotron Rad. 16, 143–153

Owen, R.L. — 2009-09-01

Current events

2009-07-01

Current events

2009-05-01

Performance of single-photon-counting PILATUS detector modules

Kraft, P. — 2009-05-01

PILATUS is a silicon hybrid pixel detector system, operating in single-photon-counting mode, that has been developed at the Paul Scherrer Institut for the needs of macromolecular crystallography at the Swiss Light Source (SLS). A calibrated PILATUS module has been characterized with monochromatic synchrotron radiation. The influence of charge sharing on the count rate and the overall energy resolution of the detector were investigated. The dead-time of the system was determined using the attenuated direct synchrotron beam. A single module detector was also tested in surface diffraction experiments at the SLS, whereby its performance regarding fluorescence suppression and saturation tolerance were evaluated, and have shown to greatly improve the sensitivity, reliability and speed of surface diffraction data acquisition.

X-ray diffractometry for the structure determination of a submicrometre single powder grain

Yasuda, N. — 2009-05-01

A high-precision diffractometer has been developed for the structure analysis of a submicrometre-scale single grain of a powder sample at the SPring-8 BL40XU undulator beamline. The key design concept is the combination of a stable focused synchrotron radiation beam and the precise axis control of the diffractometer, which allows accurate diffraction intensity data of a submicrometre-scale single powder grain to be measured. The phase zone plate was designed to create a high-flux focused synchrotron radiation beam. A low-eccentric goniometer and high-precision sample positioning stages were adopted to ensure the alignment of a micrometre-scale focused synchrotron radiation beam onto the submicrometre-scale single powder grain. In order to verify the diffractometer performance, the diffraction pattern data of several powder grains of BaTiO3, of dimensions ∼600 × 600 × 300 nm, were measured. By identifying the diffraction data set of one single powder grain, the crystal structure was successfully determined with a reliable factor of 5.24%.

100 ps time-resolved solution scattering utilizing a wide-bandwidth X-ray beam from multilayer optics

Ichiyanagi, K. — 2009-05-01

100 ps time-resolved X-ray solution-scattering capabilities have been developed using multilayer optics at the beamline NW14A, Photon Factory Advanced Ring, KEK. X-ray pulses with an energy bandwidth of ΔE/E = 1–5% are generated by reflecting X-ray pulses (ΔE/E = 15%) through multilayer optics, made of W/B4C or depth-graded Ru/C on silicon substrate. This tailor-made wide-bandwidth X-ray pulse provides high-quality solution-scattering data for obtaining photo-induced molecular reaction dynamics. The time-resolved solution scattering of CH2I2 in methanol is demonstrated as a typical example.

Comments on Migration of mercury from dental amalgam through human teeth by H. H. Harris et al. (2008). J. Synchrotron Rad. 15, 123–128

Guzzi, G. — 2009-05-01

Radiation damage in protein crystals examined under various conditions by different methods

Garman, E.F. — 2009-03-01

Investigation of radiation damage in protein crystals has progressed in several directions over the past couple of years. There have been improvements in the basic procedures such as calibration of the incident X-ray intensity and calculation of the dose likely to be deposited in a crystal of known size and composition with this intensity. There has been increased emphasis on using additional techniques such as optical, Raman or X-ray spectroscopy to complement X-ray diffraction. Apparent discrepancies between the results of different techniques can be explained by the fact that they are sensitive to different length scales or to changes in the electronic state rather than to movement of atoms. Investigations have been carried out at room temperature as well as cryo-temperatures and, in both cases, with the introduction of potential scavenger molecules. These and other studies are leading to an overall description of the changes which can occur when a protein crystal is irradiated with X-rays at both cryo- and room temperatures. Results from crystallographic and spectroscopic radiation-damage experiments can be reconciled with other studies in the field of radiation physics and chemistry.

A beginner's guide to radiation damage

Holton, J.M. — 2009-03-01

Many advances in the understanding of radiation damage to protein crystals, particularly at cryogenic temperatures, have been made in recent years, but with this comes an expanding literature, and, to the new breed of protein crystallographer who is not really interested in X-ray physics or radiation chemistry but just wants to solve a biologically relevant structure, the technical nature and breadth of this literature can be daunting. The purpose of this paper is to serve as a rough guide to radiation damage issues, and to provide references to the more exacting and detailed work. No attempt has been made to report precise numbers (a factor of two is considered satisfactory), and, since there are aspects of radiation damage that are demonstrably unpredictable, the `worst case scenario' as well as the `average crystal' are discussed in terms of the practicalities of data collection.

Response to Guzzi & Pigatto's Comments on Migration of mercury from dental amalgam through human teeth by H. H. Harris et al. (2008). J. Synchrotron Rad. 15, 123–128

Harris, H.H. — 2009-05-01

Synchrotron-based radioscopy employing spatio-temporal micro-resolution for studying fast phenomena in liquid metal foams

Rack, A. — 2009-05-01

Investigations of pore coalescence and individual cell wall collapse in an expanding liquid metal foam by means of X-ray radioscopy with spatio-temporal micro-resolution are reported. By using white synchrotron radiation for imaging, the rupture of a film and the subsequent merger of two neighbouring bubbles could be recorded with a time sampling rate of 40000 frames s−1 (25 µs exposure time) and a spatial sampling rate of 20 µm. The rupture time of a cell wall was found to be in the range of 300 µs. This value is in agreement with theoretical considerations which assume an inertia-dominated rupture time of cell walls in liquid metal foams.

Colouring cryo-cooled crystals: online microspectrophotometry

McGeehan, J. — 2009-03-01

X-rays can produce a high concentration of radicals within cryo-cooled macromolecular crystals. Some radicals have large extinction coefficients in the visible (VIS) range of the electromagnetic spectrum, and can be observed optically and spectrally. An online microspectrophotometer with high temporal resolution has been constructed that is capable of measuring UV/VIS absorption spectra (200–1100 nm) during X-ray data collection. The typical X-ray-induced blue colour that is characteristic of a wide range of cryo-conditions has been identified as trapped solvated electrons. Disulphide-containing proteins are shown to form disulphide radicals at millimolar concentrations, with absorption maxima around 400 nm. The solvated electrons and the disulphide radicals seem to have a lifetime in the range of seconds up to minutes at 100 K. The temperature dependence of the kinetics of X-ray-induced radical formation is different for the solvated electrons compared with the disulphide radicals. The online microspectrophotometer provides a technique complementary to X-ray diffraction for analysing and characterizing intermediates and redox states of proteins and enzymes.

Current events

2009-01-01

Current events

2009-03-01

A new on-axis multimode spectrometer for the macromolecular crystallography beamlines of the Swiss Light Source

Owen, R.L. — 2009-03-01

X-ray crystallography at third-generation synchrotron sources permits tremendous insight into the three-dimensional structure of macromolecules. Additional information is, however, often required to aid the transition from structure to function. In situ spectroscopic methods such as UV–Vis absorption and (resonance) Raman can provide this, and can also provide a means of detecting X-ray-induced changes. Here, preliminary results are introduced from an on-axis UV–Vis absorption and Raman multimode spectrometer currently being integrated into the beamline environment at X10SA of the Swiss Light Source. The continuing development of the spectrometer is also outlined.

Determination of X-ray flux using silicon pin diodes

Owen, R.L. — 2009-03-01

Accurate measurement of photon flux from an X-ray source, a parameter required to calculate the dose absorbed by the sample, is not yet routinely available at macromolecular crystallography beamlines. The development of a model for determining the photon flux incident on pin diodes is described here, and has been tested on the macromolecular crystallography beamlines at both the Swiss Light Source, Villigen, Switzerland, and the Advanced Light Source, Berkeley, USA, at energies between 4 and 18 keV. These experiments have shown that a simple model based on energy deposition in silicon is sufficient for determining the flux incident on high-quality silicon pin diodes. The derivation and validation of this model is presented, and a web-based tool for the use of the macromolecular crystallography and wider synchrotron community is introduced.

Capturing molecular structural dynamics by 100 ps time-resolved X-ray absorption spectroscopy

Sato, T. — 2009-01-01

An experimental set-up for time-resolved X-ray absorption spectroscopy with 100 ps time resolution at beamline NW14A at the Photon Factory Advanced Ring is presented. The X-ray positional active feedback to crystals in a monochromator combined with a figure-of-merit scan of the laser beam position has been utilized as an essential tool to stabilize the spatial overlap of the X-ray and laser beams at the sample position. As a typical example, a time-resolved XAFS measurement of a photo-induced spin crossover reaction of the tris(1,10-phenanthrorine)iron(II) complex in water is presented.

Current events

2008-11-01

Current events

2008-09-01

PI-MTE cameras now twice as fast with 2 MHz readout

Flint-Baum, D. — 2008-09-01

A coherent synchrotron X-ray microradiology investigation of bubble and droplet coalescence

Weon, B.M. — 2008-11-01

A quantitative application of microradiology with coherent X-rays to the real-time study of microbubble and microdroplet coalescence phenomena, with specific emphasis on the size relations in three-body events, is presented. The results illustrate the remarkable effectiveness of coherent X-ray imaging in delineating interfaces in multiphase systems, in accurately measuring their geometric properties and in monitoring their dynamics.

Current events

2008-07-01

Current events

2008-05-01

Development of an X-ray HARP–FEA detector system for high-throughput protein crystallography

Miyoshi, T. — 2008-05-01

A new detector system for protein crystallography is now being developed based on an X-ray HARP–FEA (high-gain avalanche rushing amorphous photoconductor–field emitter array), which consists of an amorphous selenium membrane and a matrix field emitter array. The combination of the membrane avalanche effect with a single driven FEA has several advantages over currently available area detectors, including higher sensitivity, higher spatial resolution and a higher frame rate. Preliminary evaluation of the detector has been carried out and its effectiveness has been confirmed. Next, diffraction images were measured with continuous rotation of a protein crystal, and the images were compared with those measured by the existing CCD detector; the system successfully obtained high-spatial-resolution images. Using shutterless measurement, the total measurement time can be reduced significantly, making the method appropriate for high-throughput protein crystallography. The X-ray HARP–FEA detector is an attractive candidate for the next generation of X-ray area detectors.

Structure of six-transmembrane cation channels revealed by single-particle analysis from electron microscopic images

Mio, K. — 2008-05-01

Six-transmembrane (6-TM) cation channels are plasma membrane-integral components of cellular signaling pathways conserved in almost all species, including animals, plants and some kinds of prokaryotes. These channels selectively permeate cations in response to various signals. In excitable and non-excitable mammalian cells, 6-TM cation channels play fundamental roles, including the generation of action potential and its transmission, the regulation of intracellular ion concentrations, and the activation of signaling cascades by humoral or mechanical pathways. Recently, the structures of three different 6-TM-type cation channels have been determined using single-particle analysis from cryo-electron microscopy images: the voltage-sensitive sodium channel, the IP3 receptor and the TRPC3 channel. The basic structure of the molecules is similar: a bell-like shape comprising a relatively small extracellular (or luminal) domain, a protein-dense transmembrane domain and an expanded cytoplasmic domain. However, in detail, the cytoplasmic architectures are different from one another and are diversely evolved to their specific physiological functions.

Mutagenesis of the crystal contact of acidic fibroblast growth factor

Honjo, E. — 2008-05-01

An attempt has been made to improve a crystal contact of human acidic fibroblast growth factor (haFGF; 140 amino acids) to control the crystal growth, because haFGF crystallizes only as a thin-plate form, yielding crystals suitable for X-ray but not neutron diffraction. X-ray crystal analysis of haFGF showed that the Glu81 side chain, located at a crystal contact between haFGF molecules, is in close proximity with an identical residue related by crystallographic symmetry, suggesting that charge repulsion may disrupt suitable crystal-packing interactions. To investigate whether the Glu residue affects the crystal-packing interactions, haFGF mutants in which Glu81 was replaced by Ala, Val, Leu, Ser and Thr were constructed. Although crystals of the Ala and Leu mutants were grown as a thin-plate form by the same precipitant (formate) as the wild type, crystals of the Ser and Thr mutants were grown with increased thickness, yielding a larger overall crystal volume. X-ray structural analysis of the Ser mutant determined at 1.35 Å resolution revealed that the hydroxy groups of Ser are linked by hydrogen bonds mediated by the formate used as a precipitant. This approach to engineering crystal contacts may contribute to the development of large protein crystals for neutron crystallography.

Current events

2008-03-01

Highly bright X-ray generator using heat of fusion with a specially designed rotating anticathode

Sakabe, N. — 2008-05-01

A new type of rotating anticathode X-ray generator has been developed, in which the electron beam irradiates the inner surface of a U-shaped anticathode (Cu). A high-flux electron beam is focused on the inner surface by optimizing the shape of the bending magnet. The power of the electron beam can be increased to the point at which the irradiated part of the inner surface is melted, because a strong centrifugal force fixes the melted part on the inner surface. When the irradiated part is melted, a large amount of energy is stored as the heat of fusion, resulting in emission of X-rays 4.3 times more brilliant than can be attained by a conventional rotating anticathode. Oscillating translation of the irradiated position on the inner surface during use is expected to be very advantageous for extending the target life. A carbon film coating on the inner surface is considered to suppress evaporation of the target metal and will be an important technique in further realization of highly bright X-ray generation.

Implementation of remote monitoring and diffraction evaluation systems at the Photon Factory macromolecular crystallography beamlines

Yamada, Y. — 2008-05-01

Owing to recent advances in high-throughput technology in macromolecular crystallography beamlines, such as high-brilliant X-ray sources, high-speed readout detectors and robotics, the number of samples that can be examined in a single visit to the beamline has increased dramatically. In order to make these experiments more efficient, two functions, remote monitoring and diffraction image evaluation, have been implemented in the macromolecular crystallography beamlines at the Photon Factory (PF). Remote monitoring allows scientists to participate in the experiment by watching from their laboratories, without having to come to the beamline. Diffraction image evaluation makes experiments easier, especially when using the sample exchange robot. To implement these two functions, two independent clients have been developed that work specifically for remote monitoring and diffraction image evaluation. In the macromolecular crystallography beamlines at PF, beamline control is performed using STARS (simple transmission and retrieval system). The system adopts a client–server style in which client programs communicate with each other through a server process using the STARS protocol. This is an advantage of the extension of the system; implementation of these new functions required few modifications of the existing system.

High-resolution X-ray microdiffraction analysis of natural teeth

Xue, J. — 2008-05-01

The main component of natural teeth was determined many years ago as calcium phosphate, mostly in the form of hydroxyapatite with different crystallites. In the past, the method used in tooth crystal investigation has been mainly powder X-ray diffraction analysis, but this method has its drawbacks, i.e. the destruction of the natural tooth structure and the difficulty in examining the preferred orientation in different layers of the tooth. During the last century, microzone X-ray diffraction on the tooth surface was carried out, but, as the technology was less sophisticated, the results obtained were not very detailed. The newly developed microdiffraction equipment permits analysis of the microzone of teeth in situ. To test this new microdiffraction equipment, microdiffraction analysis of one natural healthy deciduous molar tooth and one carious deciduous molar tooth has been performed, using a Bruker D8 instrument. Phase analysis of the two teeth was performed; the crystal size at six test points in the natural healthy tooth was calculated by reflection (211), and the crystal preferred orientation of reflection (300) and reflection (002) at six test points in the natural healthy tooth were compared. The results showed that the tooth was a kind of biological mixed crystal composed of several crystal phases, the main crystal phase being hydroxyapatite. The crystal size grew larger going from the dentin to the enamel. The crystal preferred orientation mainly existed in the enamel, especially in the reflection (002). From our experiment, layer orientation and continuous crystal variations in teeth could be conveniently studied using fast online measurements by high-resolution X-ray microdiffraction equipment.

X-ray crystallographic and biochemical characterizations of a mutant photosystem II complex from Thermosynechococcus vulcanus with the psbTc gene inactivated by an insertion mutation

Henmi, T. — 2008-05-01

The crystal structure of a photosystem II (PSII) dimer from Thermosynechococcus vulcanus with its psbTc gene inactivated by insertion mutation of an antibiotic cassette in a site in the C-terminal region was analyzed at 3.8 Å resolution. In the crystal structure of the mutant PSII, the transmembrane helix of PsbTc remains, whereas the C-terminal loop of PsbTc has disappeared. In addition, the PsbM subunit, which seemed to be lost in a PsbTc-deletion mutant PSII of T. elongatus, is still present. The deletion of the C-terminal loop of PsbTc in the mutant PSII was verified by mass spectrometry. Thus, the insertion mutation of psbTc eliminated only the C-terminal loop of this subunit. Nevertheless, some features of the mutant PSII, namely a destabilization of the dimeric form and a slight decrease of the oxygen-evolving activity, were observed in the mutant, indicating that the C-terminal loop of PsbTc functions to maintain the stability of the PSII dimer and the activity of oxygen evolution.

Protein crystallization in a 100 nl solution with new stirring equipment

Maki, S. — 2008-05-01

To investigate quantitatively the effects of stirring on protein crystallization, a new stirring system which can agitate a protein solution, ∼100 nl, by providing Hagen–Poiseuille flow has been successfully developed. In addition, this new stirring system provides flow with a well defined pattern and velocity. Using this system, hen egg-white lysozyme was crystallized in 100–200 nl solutions while being stirred. The optimum stirring conditions for lysozyme crystals have been explored by evaluating the Reynolds (Re) number and the crystals obtained. Intermittent flow, as well as a low Re number, was found to contribute significantly to the growth of a smaller number of larger crystals.

Structural studies on Helicobacter pylori ATP-dependent protease, FtsH

Kim, S.H. — 2008-05-01

The ATP-dependent protease, FtsH, degrades misassembled membrane proteins for quality control like SecY, subunit a of FoF1-ATPase, and YccA, and digests short-lived soluble proteins in order to control their cellular regulation, including σ32, LpxC and λcII. The FtsH protein has an N-terminal transmembrane segment and a large cytosolic region that consists of two domains, an ATPase and a protease domain. To provide a structural basis for the nucleotide-dependent domain motions and a better understanding of substrate translocation, the crystal structures of the Helicobacter pylori (Hp) FtsH ATPase domain in the nucleotide-free state and complexed with ADP, were determined. Two different structures of HpFtsH ATPase were observed, with the nucleotide-free state in an asymmetric unit, and these structures reveal the new forms and show other conformational differences between the nucleotide-free and ADP-bound state compared with previous structures. In particular, one HpFtsH Apo structure has a considerable rotation difference compared with the HpFtsH ADP complex, and this large conformational change reveals that FtsH may have the mechanical force needed for substrate translocation.

Protein structures by spallation neutron crystallography

Langan, P. — 2008-05-01

The Protein Crystallography Station at Los Alamos Neutron Science Center is a high-performance beamline that forms the core of a capability for neutron macromolecular structure and function determination. This capability also includes the Macromolecular Neutron Crystallography (MNC) consortium between Los Alamos (LANL) and Lawrence Berkeley National Laboratories for developing computational tools for neutron protein crystallography, a biological deuteration laboratory, the National Stable Isotope Production Facility, and an MNC drug design consortium between LANL and Case Western Reserve University.

Science experiments via telepresence at a synchrotron radiation source facility

Warren, J.E. — 2008-03-01

Station 9.8 is one of the most oversubscribed and high-throughput stations at the Synchrotron Radiation Source, Daresbury, whereby awarded experimental time is limited, data collections last normally no longer than an hour, user changeover is normally every 24 h, and familiarity with the station systems can be low. Therefore time lost owing to technical failures on the station has a dramatic impact on productivity. To provide 24 h support, the application of a turnkey communication system has been implemented, and is described along with additional applications including its use for inter-continental classroom instruction, user training and remote participation.

Ultrahigh-resolution crystallography and related electron density and electrostatic properties in proteins

Lecomte, C. — 2008-05-01

With an increasing number of biological macromolecular crystal structures measured at ultrahigh resolution (1 Å or better), it is necessary to extend to large systems the experimental valence electron density modelling that is applied to small molecules. A database of average multipole populations has been built, describing the electron density of chemical groups in all 20 amino acids found in proteins. It allows calculation of atomic aspherical scattering factors, which are the starting point for refinement of the protein electron density, using the MoPro software. It is shown that the use of non-spherical scattering factors has a major impact on crystallographic statistics and results in a more accurate crystal structure, notably in terms of thermal displacement parameters and bond distances involving H atoms. It is also possible to obtain a realistic valence electron density model, which is used in the calculation of the electrostatic potential and energetic properties of proteins.

Interaction of HIV-1 aspartic protease with its inhibitor, by molecular dynamics and ab initio fragment molecular orbital method

Koyano, K. — 2008-05-01

For the three complex crystal structures of HIV-1 aspartic protease (an enzyme of AIDS) with its inhibitor in the Protein Data Bank, molecular dynamics of the generalized Born surface area and the ab initio fragment molecular orbital of an ABINIT-MP calculation was performed to obtain the binding free energy, the molecular orbital energy, the interaction energy of residues with an inhibitor and the charge transfer at the active site. The inhibitors are five symmetric cyclic ureas, of which three were modelled, and an asymmetric dipeptide. The interaction energy of the inhibitor at the active sites of aspartic acid is as great as 50 kcal mol−1, coinciding with a tetrahedral transition state. For the inhibitor with a higher affinity, charge was transferred to the inhibitor from the active site. The difference in symmetry of the inhibitor was not evident. Binding free energy corresponds to the experimental value of the binding constant, while molecular orbital energy does not always, which is considered to be an entropy effect.

Effect of amino acids and amino acid derivatives on crystallization of hemoglobin and ribonuclease A

Ito, L. — 2008-05-01

Determination of the appropriate conditions for protein crystallization remains a highly empirical process. Preventing protein aggregation is necessary for the formation of single crystals under aggregation-prone solution conditions. Because many amino acids and amino acid derivatives offer a unique combination of solubility and stabilizing properties, they open new avenues into the field of protein aggregation research. The use of amino acids and amino acid derivatives can potentially influence processes such as heat treatment and refolding reactions. The effect of the addition of several amino acids, such as lysine, and several amino acid derivatives, such as glycine ethyl ester and glycine amide, on the crystallization of equine hemoglobin and bovine pancreatic ribonuclease A has been examined. The addition of these amino acids and amino acid derivatives expanded the range of precipitant concentration in which crystals formed without aggregation. The addition of such additives appears to promote the crystallization of proteins.

Novel dimer structure of a membrane-bound protease with a catalytic Ser–Lys dyad and its linkage to stomatin

Yokoyama, H. — 2008-05-01

Membrane-bound proteases are involved in various regulatory functions. A previous report indicates that the N-terminal region of PH1510 (1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii is a serine protease with a catalytic Ser–Lys dyad (Ser97 and Lys138), and specifically cleaves the C-terminal hydrophobic region of the p-stomatin PH1511. According to the crystal structure of the wild-type 1510-N in dimeric form, the active site around Ser97 is in a hydrophobic environment suitable for the hydrophobic substrates. This article reports the crystal structure of the K138A mutant of 1510-N at 2.3 Å resolution. The determined structure contains one molecule per asymmetric unit, but 1510-N is active in dimeric form. Two possible sets of dimer were found from the symmetry-related molecules. One dimer is almost the same as the wild-type 1510-N. Another dimer is probably in an inactive form. The L2 loop, which is disordered in the wild-type structure, is significantly kinked at around A-138 in the K138A mutant. Thus Lys138 probably has an important role on the conformation of L2.

X-ray beam stabilization at BL-17A, the protein microcrystallography beamline of the Photon Factory

Igarashi, N. — 2008-05-01

BL-17A is a new structural biology beamline at the Photon Factory, Japan. The high-brilliance beam, derived from the new short-gap undulator (SGU#17), allows for unique protein crystallographic experiments such as data collection from microcrystals and structural determination using softer X-rays. However, microcrystal experiments require robust beam stability during data collection and minor fluctuations could not be ignored. Initially, significant beam instability was observed at BL-17A. The causes of the beam instability were investigated and its various sources identified. Subsequently, several effective countermeasures have been implemented, and the fluctuation of the beam intensity successfully suppressed to within 1%. Here the instability reduction techniques used at BL-17A are presented.

Performance of an electron gun for a high-brightness X-ray generator

Sugimura, T. — 2008-05-01

A prototype thermionic electron gun for a high-brightness X-ray generator has been developed. Its extraction voltage and design current are 60 kV and 100 mA (DC), respectively. The X-ray generator aims towards a maximum brilliance of 60 kW mm−2. The beam sizes at the rotating anticathode must therefore be within 1.0 mm × 0.1 mm and a small beam emittance is required. The fabricated electron gun optimizes an aperture grid and a Whenelt electrode. The performance of the prototype electron gun measured using pulsed-beam tests is as follows: maximum beam current, 85.7 mA; beam focus size at the rotating anticathode, 0.79 mm × 0.13 mm. In DC beam tests, FWHM beam sizes were measured to be 0.65 mm × 0.08 mm at the rotating anticathode with a beam current of 45 mA. The beam current recently reached ∼60 mA with some thermal problems.

Current events

2008-01-01

Solution structures of RseA and its complex with RseB

Jin, K.S. — 2008-05-01

The bacterial envelope stress response, which is responsible for sensing stress signals in the envelope and for turning on the σE-dependent transcription, is modulated by the binding of RseB to RseA. In this study, the solution structures of RseA and its complex with RseB were analyzed using circular dichroism and small-angle X-ray scattering. The periplasmic domain of RseA is unstructured and flexible when it is not bound to RseB. However, upon the formation of the stable complex with RseB, RseA induces conformational changes in RseB and, at the same time, RseA becomes more structured. Furthermore, it appears that some other undefined region of RseA, as well as the previously identified minimum region (amino acid 169–186), is also involved in RseB binding. It is thought that these conformational changes are relevant to the proteolytic cleavage of RseA and the modulation of envelope stress response.

Protein disulfide bond generation in Escherichia coli DsbB–DsbA

Inaba, K. — 2008-05-01

Protein disulfide bond formation is catalyzed by a series of Dsb enzymes present in the periplasm of Escherichia coli. The crystal structure of the DsbB–DsbA–ubiquinone ternary complex provided important insights into mechanisms of the de novo disulfide bond generation cooperated by DsbB and ubiquinone and of the disulfide bond shuttle from DsbB to DsbA. The structural basis for prevention of the crosstalk between the DsbA–DsbB oxidative and the DsbC–DsbD reductive pathways has also been proposed.

Fragment approaches in structure-based drug discovery

Hubbard, R.E. — 2008-05-01

There has been considerable interest recently in what is known as `fragment-based lead discovery'. The novel feature of the approach is to begin with small low-affinity compounds. The main advantage is that a larger potential chemical diversity can be sampled with fewer compounds, which is particularly important for new target classes. The approach relies on careful design of the fragment library, a method that can detect binding of the fragment to the protein target, determination of the structure of the fragment bound to the target, and the conventional use of structural information to guide compound optimization. In this article the methods are reviewed, and experiences in fragment-based discovery of lead series of compounds against kinases such as PDK1 and ATPases such as Hsp90 are discussed. The examples illustrate some of the key benefits and issues of the approach and also provide anecdotal examples of the patterns seen in selectivity and the binding mode of fragments across different protein targets.

Crystal twinning of human MD-2 recognizing endotoxin cores of lipopolysaccharide

Ohto, U. — 2008-05-01

Twinning of crystals causes overlapping of two or more reciprocal lattice points, and hence structure amplitudes for a single crystalline domain are hardly obtained from X-ray diffraction intensities. MD-2 protein forms a stable complex with Toll-like receptor 4 and recognizes bacterial lipopolysaccharide (LPS). Excessive immune responses activated by LPS cause septic shocks. Saccharide-trimmed human MD-2 crystallizes in the tetragonal form with apparent Laue symmetry of 4/mmm, and diffraction intensities from these crystals indicate crystal twinning. The crystal consists of two different domains, A and B. The cA axis of domain A coincides with the cB axis of domain B with a smaller lattice, and the aA axis corresponds to the (aB + bB) axis. This twinning severely imposes difficulty in structure determination. Through optimization of cryoprotectant, domain A was thoroughly transformed into domain B. The crystal containing only domain B is in space group P41212 with one MD-2 molecule in the asymmetric unit. The structure of this form of MD-2 as well as its complex with antiendotoxic lipid IVa was successfully determined using the multiple isomorphous replacement method.

An optimal exposure strategy for cryoprotected virus crystals with lattice constants greater than 1000 Å

Gan, L. — 2008-05-01

Studies of icosahedral virus capsids provide insights into the function of supramolecular machines. Virus capsid crystals have exceptionally large unit cells; as a result, they diffract weakly compared with protein crystals. HK97 is a dsDNA lambda-like bacteriophage whose 13 MDa capsid expands from 550 Å to 650 Å with large subunit conformational changes during virus maturation. The HK97 penultimate maturation intermediate was crystallized in a tetragonal unit cell that has lattice constants of 1010 Å × 1010 Å × 730 Å. The crystals could be cryoprotected, but diffracted to a modest resolution of 5 Å at a bending-magnet beamline. When these crystals were optimally exposed with two orders-of-magnitude more photons from a new insertion-device beamline, data extending to better than 3.8 Å resolution were obtained. Here, the strategies to collect and process such data are described. These strategies can be adapted for other crystals with large unit cells and for microcrystals.

Mail-in data collection at SPring-8 protein crystallography beamlines

Okazaki, N. — 2008-05-01

A mail-in data collection system makes it possible for beamline users to collect diffraction data without visiting a synchrotron facility. In the mail-in data collection system at SPring-8, users pack crystals into sample trays and send the trays to SPring-8 via a courier service as the first step. Next, the user specifies measurement conditions and checks the diffraction images via the Internet. The user can also collect diffraction data using an automated sample changer robot and beamline control software. For distant users there is a newly developed data management system, D-Cha. D-Cha provides a graphical user interface that enables the user to specify the experimental conditions for samples and to check and download the diffraction images using a web browser. This system is now in routine operation and is contributing to high-throughput beamline operation.

High-throughput operation of sample-exchange robots with double tongs at the Photon Factory beamlines

Hiraki, M. — 2008-05-01

Sample-exchange robots that can exchange cryo-pins bearing protein crystals out of experimental hutches according to user instructions have been developed. The robots were designed based on the SAM (Stanford Synchrotron Research Laboratory automated mounting) system. In order to reduce the time required for the sample exchange, the single tongs of the SAM system were modified and a double-tongs system that can hold two cryo-pins at the same time was developed. Robots with double tongs can move to the goniometer head holding the next cryo-pin with one set of tongs, dismount the experimented cryo-pin with the other set, and then mount the next pin onto the goniometer head without leaving the diffractometer area. Two different types of tongs have been installed: single tongs at beamlines BL-5A and AR-NW12A, and a double-tongs system at beamline BL-17A of the Photon Factory. The same graphical user interface software for operation of the sample-exchange robots is used at all beamlines, however, so that users do not need to consider differences between the systems. In a trial, the robot with double tongs could exchange samples within 10 s.

Deduced catalytic mechanism of d-amino acid amidase from Ochrobactrum anthropi SV3

Okazaki, S. — 2008-05-01

d-Amino acid amidase (DAA) from Ochrobactrum anthropi SV3 catalyzes d-stereospecific hydrolysis of amino acid amides. DAA has attracted attention as a catalyst for the stereospecific production of d-amino acids, although the mechanism that drives the reaction has not been clear. Previously, the structure of DAA was classified into two types, a substrate-bound state with an ordered Ω loop, and a ground state with a disordered Ω loop. Because the binding of the substrate facilitates ordering, this transition was regarded to be induced fit motion. The angles and distances of hydrogen bonds at Tyr149 Oη, Ser60 Oγ and Lys63 Nζ revealed that Tyr149 Oη donates an H atom to a water molecule in the substrate-bound state, and that Tyr149 Oη donates an H atom to Ser60 Oγ or Lys63 Nζ in the ground state. Taking into consideration the locations of the H atoms of Tyr149 Oη, Ser60 Oγ and Lys63 Nζ, a catalytic mechanism of DAA activity is presented, wherein a shift of an H atom at Tyr149 Oη in the substrate-bound versus the ground state plays a significant role in the reaction. This mechanism explains well why acylation proceeds and deacylation does not proceed in the substrate-bound state.

Key steps in the structure-based optimization of the hepatitis C virus NS3/4A protease inhibitor SCH503034

Madison, V. — 2008-05-01

The structures of both native and S139A holo-HCV NS3/4A protease domain were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contributions to the binding energy arise from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease, which is currently in clinical trials.

A degradation signal recognition in prokaryotes

Park, E.Y. — 2008-05-01

The degradation of ssrA-tagged substrates in prokaryotes is conducted by a subset of ATP-dependent proteases, including ClpXP complex. More than 630 sequences of ssrA have been identified from 514 species, and are conserved in a wide range of prokaryotes. SspB protein markedly stimulates the degradation of these ssrA-tagged substrates by the ClpXP proteolytic machine. The dimeric SspB protein is composed of a compact ssrA-binding domain, which has a dimerization surface and a flexible C-terminal tail with a ClpX-binding motif at its very end. Since SspB is an adaptor protein for the ClpXP complex, designed mutagenesis, fluorescence spectroscopy, biochemistry and X-ray crystallography have been used to investigate the mechanism of delivery of ssrA-tagged proteins. In this paper the structural basis of ssrA-tag recognition by ClpX and SspB, as well as SspB-tail recognition by ZBD, is described.

Complex of human neutrophil elastase with 1/2SLPI

Koizumi, M. — 2008-05-01

SLPI (secretory leukocyte protease inhibitor) is a 107-residue non-glycosylated protease inhibitor, which inhibits a wide range of serine proteases, trypsin, chymotrypsin, neutrophil elastase, chymase and cathepsin G. X-ray crystallographic analyses have shown that SLPI comprises two separate domains of similar architecture [Grütter, Fendrich, Huber & Bode (1988), EMBO J. 7, 345–351] and the C-terminal domain interacts with bovine α-chymotrypsin. In order to understand SLPI's multiple functions against various serine proteases, the complex HNE (human neutrophil elastase) has been co-crystallized with 1/2SLPI (recombinant C-terminal domain of SLPI; Arg58–Ala107), which has a biological activity similar to full SLPI. The 1/2SLPI and HNE complex structure was solved at 1.7 Å resolution, and compared with the interaction mechanism of elafin, which is a specific inhibitor of elastase. It was found that P1 Leu72i and six hydrogen bonds between the main chains in the primary contact region have sufficient ability to inhibit HNE and PPE (porcine pancreatic elastase), and P5 Tyr68i is important in increasing the selectivity of 1/2SLPI against HNE. The mechanisms of the functions of SLPI are relatively unknown, but the current study could help understand the selectivity of SLPI against HNE and PPE.

Crystallization and evaluation of hen egg-white lysozyme crystals for protein pH titration in the crystalline state

Iwai, W. — 2008-05-01

To observe the ionized status of the amino acid residues in proteins at different pH (protein pH titration in the crystalline state) by neutron diffraction, hen egg-white lysozyme was crystallized over a wide pH range (2.5–8.0). Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined. At pH < 4.5 the border between the metastable region and the nucleation region shifted to the left (lower precipitant concentration) in the phase diagram, and at pH > 4.5 the border shifted to the right (higher precipitant concentration). The qualities of these crystals were characterized using the Wilson plot method. The qualities of all crystals at different pH were more or less equivalent (B-factor values within 25–40). It is expected that neutron diffraction analysis of these crystals of different pH provides equivalent data in quality for discussions of protein pH titration in the crystalline state of hen egg-white lysozyme.

Hydrogen bonds of DsrD protein revealed by neutron crystallography

Chatake, T. — 2008-05-01

The features of hydrogen bonds in DsrD protein from sulfate-reducing bacteria have been investigated by neutron protein crystallography. The function of DsrD has not yet been elucidated clearly, but its X-ray crystal structure revealed that it comprises a winged-helix motif and shows the highest structural homology to the DNA-binding proteins. Since any neutron structure of a DNA recognition protein has not yet been obtained, here detailed information on the hydrogen bonds in the winged-helix-motif protein is given and the following features found. (i) The number of hydrogen bonds per amino acid of DsrD is relatively fewer than for other proteins for which neutron structures were determined previously. (ii) Hydrogen bonds are localized between main-chain and main-chain atoms; there are few hydrogen bonds between main-chain and side-chain atoms and between side-chain and side-chain atoms. (iii) Hydrogen bonds inducted by protonation of specific amino acid residues (Glu50) seem to play an essential role in the dimerization of DsrD. The former two points are related to the function of the DNA-binding protein; the three-dimensional structure was mainly constructed by hydrogen bonds in main chains, while the side chains appeared to be used for another role. The latter point would be expected to contribute to the crystal growth of DsrD.

Cost-effective upgrade of a focusing system for inelastic X-ray scattering experiments under high pressure

Huang, C.-Y. — 2008-01-01

Inelastic X-ray scattering (IXS) is a powerful technique capable of probing the dynamic behavior and electronic structure of materials. For IXS experiments under high pressure up to the megabar range using state-of-the-art diamond-anvil-cell technology, the sample volume is limited to the order of 1 × 10−3 mm3 for which a beam focus of the same order and less is often required. In this paper a scheme utilizing a set of low-cost and compact Kirkpatrick–Baez mirrors for upgrading the existing optical system of the Taiwan IXS beamline at SPring-8 is described. The scheme as implemented improves the focus to 13 µm × 16 µm (horizontal × vertical) with a transmission of up to 72% and a flux density gain of over 30 times, which has enhanced substantially the efficiency of the beamline for high-pressure research.

Trimeric structure and conformational equilibrium of M-ficolin fibrinogen-like domain

Tanio, M. — 2008-05-01

Ficolins are pathogen-recognition molecules in innate immune systems. The crystal structure of the human M-ficolin recognition domain (FD1) has been determined at 1.9 Å resolution, and compared with that of the human fibrinogen γ fragment, tachylectin-5A, L-ficolin and H-ficolin. The overall structure of FD1 is similar to that of the other proteins, although the peptide bond between Asp282 and Cys283, which is in a predicted ligand-binding site, is a normal trans bond, unlike the cases of the other proteins. Analysis of the pH-dependent ligand-binding activity of FD1 in solution suggested that a conformational equilibrium between active and non-active forms in the ligand-binding region, involving cis-trans isomerization of the Asp282—Cys283 peptide bond, contributes to the discrimination between self and non-self, and that the pKa values of His284 are 6.1 and 6.3 in the active and non-active forms, respectively.

Crystallization of the Atg12–Atg5 conjugate bound to Atg16 by the free-interface diffusion method

Noda, N.N. — 2008-05-01

Autophagy mediates the bulk degradation of cytoplasmic components in lysosomes/vacuoles. Five autophagy-related (Atg) proteins are involved in a ubiquitin-like protein conjugation system. Atg12 is conjugated to its sole target, Atg5, by two enzymes, Atg7 and Atg10. The Atg12–Atg5 conjugates form a multimeric complex with Atg16. Formation of the Atg12–Atg5–Atg16 ternary complex is crucial for the functions of these proteins on autophagy. Here, the expression, purification and crystallization of the Atg12–Atg5 conjugate bound to the N-terminal region of Atg16 (Atg16N) are reported. The Atg12–Atg5 conjugates were formed by co-expressing Atg5, Atg7, Atg10 and Atg12 in Eschericia coli. The Atg12–Atg5–Atg16N ternary complex was formed by mixing purified Atg12–Atg5 conjugates and Atg16N, and was further purified by gel-filtration chromatography. Crystallization screening was performed by the free-interface diffusion method. Using obtained microcrystals as seeds, large crystals for diffraction data collection were obtained by the sitting-drop vapour-diffusion method. The crystal contained one ternary complex per asymmetric unit, and diffracted to 2.6 Å resolution.

Engines of Discovery (A Century of Particle Accelerators). By Andrew Sessler and Edmund Wilson. Pp. ix + 212. Hackensack, NJ: World Scientific Publishing 2007. Price: (hardcover) GBP 29, USD 54. ISBN 978-981-270-070-4.

Suller, V.P. — 2008-01-01

Structural insights into the similar modes of Nrf2 transcription factor recognition by the cytoplasmic repressor Keap1

Padmanabhan, B. — 2008-05-01

The cytoplasmic repressor Keap1 regulates the function of transcription factor Nrf2 which plays critical roles in oxidative and xenobiotic stresses. The Neh2 domain of Nrf2 interacts with Keap1 at the bottom region of the Kelch/β-propeller domain which is formed by double-glycine repeat and C-terminal region domains (Keap1-DC). The structure of Keap1-DC complexed with an Nrf2 peptide containing a conserved DLG motif has been determined at 1.9 Å resolution. The Keap1-bound DLG peptide possesses a hairpin conformation, and it binds to the Keap1 protein at the bottom region of the β-propeller domain. The intermolecular interaction occurs through their complementary electrostatic interactions. Comparison of the present structure with the recently reported Keap1-DC complex structure suggests that the DLG and ETGE motifs of Neh2 in Nrf2 bind to Keap1 in a similar manner but with different binding potencies.

Current events

2007-11-01

Current events

2007-09-01

Coherent X-ray scattering and lensless imaging at the European XFEL Facility

Vartanyants, I.A. — 2007-11-01

Coherent X-ray diffraction imaging is a rapidly advancing form of lensless microscopy. The phase information of the diffraction pattern is embedded in a sufficiently sampled coherent diffraction pattern. Using advanced computational methods, this diffraction pattern can be inverted to produce an image of a sample with diffraction-limited resolution. It is attractive to use high-power coherent X-ray beams produced by future X-ray free-electron lasers for imaging nanoscale condensed matter, materials and biological samples. Here, the scientific case, requirements and the possible realisation of the coherent X-ray diffraction imaging beamlines at the European XFEL Facility are presented.

Current events

2007-07-01

Developing 100 ps-resolved X-ray structural analysis capabilities on beamline NW14A at the Photon Factory Advanced Ring

Nozawa, S. — 2007-07-01

NW14A is a newly constructed undulator beamline for 100 ps time-resolved X-ray experiments at the Photon Factory Advanced Ring. This beamline was designed to conduct a wide variety of time-resolved X-ray measurements, such as time-resolved diffraction, scattering and X-ray absorption fine structure. Its versatility is allowed by various instruments, including two undulators, three diffractometers, two pulse laser systems and an X-ray chopper. The potential for the detection of structural changes on the 100 ps time scale at NW14A is demonstrated by two examples of photo-induced structural changes in an organic crystal and photodissociation in solution.

ID20: a beamline for magnetic and resonant X-ray scattering investigations under extreme conditions

Paolasini, L. — 2007-07-01

A new experimental station at ESRF beamline ID20 is presented which allows magnetic and resonant X-ray scattering experiments in the energy range 3–25 keV to be performed under extreme conditions. High magnetic field up to 10 T, high pressure up to 30 kbar combined with low temperatures down to 1.5 K are available and experiments can be performed at the M-edges of actinide elements, L-edges of lanthanides and K-edges of transition metals.

The small-angle and wide-angle X-ray scattering set-up at beamline BL9 of DELTA. Erratum

Krywka, C. — 2007-07-01

A correction is made to the equation in Krywka et al. (2007), J. Synchrotron Rad. 14, 244–251.

Current events

2007-05-01

Current events

2007-03-01

Progress in research into radiation damage in cryo-cooled macromolecular crystals

Garman, E.F. — 2007-01-01

Current events

2007-01-01

Verifying DiffEXAFS measurements with differential X-ray diffraction

Ruffoni, M.P. — 2007-01-01

Differential EXAFS (DiffEXAFS) is a novel technique for measuring atomic perturbations on a local scale. Here a complementary technique for such studies is presented: differential X-ray diffraction (DiffXRD), which may be used to independently verify DiffEXAFS results whilst using exactly the same experimental apparatus and measurement technique. A test experiment has been conducted to show that DiffXRD can be used to successfully determine the thermal expansion coefficient of SrF2.

Dose dependence of radiation damage for protein crystals studied at various X-ray energies

Shimizu, N. — 2007-01-01

Radiation damage to protein crystals is the most serious problem in obtaining accurate structures from protein crystallography. In order to examine the photon energy dependence of radiation damage, 12 to 15 data sets from each of nine tetragonal lysozyme crystals were collected at nine different X-ray energies (6.5, 7.1, 8.3, 9.9, 12.4, 16.5, 20.0, 24.8 and 33.0 keV) using beamline BL41XU at SPring-8. All results were compared on the basis of absorbed dose, expressed in Gray (Gy). Crystallographic statistics, such as the values of lattice constants, Rmerge and I/σ(I), for each data set degraded at all nine energies as the exposure time for each crystal increased. In all data sets, radiation damage was observed after the absorbed dose exceeded 106 Gy. However, from the point of view of crystallographic statistics normalized to the absorbed dose, no clear dependence on photon energy was observed in these results. Structural refinement showed that the average B-factor for the last data set was larger than that for the first data set at all energies tested. However, no energy dependence of radiation damage on B-factor was found. Furthermore, disruption of disulfide bonds due to radiation damage was observed in electron density maps even at the highest photon energy (33 keV) used in this study. Therefore, these results suggest that radiation damage in the energy range investigated could be evaluated based on absorbed dose without energy dependence, and that it is important to minimize the absorbed dose in a crystal sample for obtaining an accurate protein structure.

XANES measurements of the rate of radiation damage to selenomethionine side chains

Holton, J.M. — 2007-01-01

The radiation-induced disordering of selenomethionine (SeMet) side chains represents a significant impediment to protein structure solution. Not only does the increased B-factor of these sites result in a serious drop in phasing power, but some sites decay much faster than others in the same unit cell. These radio-labile SeMet side chains decay faster than high-order diffraction spots with dose, making it difficult to detect this kind of damage by inspection of the diffraction pattern. The selenium X-ray absorbance near-edge spectrum (XANES) from samples containing SeMet was found to change significantly after application of X-ray doses of 10–100 MGy. Most notably, the sharp `white line' feature near the canonical Se edge disappears. The change was attributed to breakage of the Cγ—Se bond in SeMet. This spectral change was used as a probe to measure the decay rate of SeMet with X-ray dose in cryo-cooled samples. Two protein crystal types and 15 solutions containing free SeMet amino acid were examined. The damage rate was influenced by the chemical and physical condition of the sample, and the half-decaying dose for the selenium XANES signal ranged from 5 to 43 MGy. These decay rates were 34- to 3.8-fold higher than the rate at which the Se atoms interacted directly with X-ray photons, so the damage mechanism must be a secondary effect. Samples that cooled to a more crystalline state generally decayed faster than samples that cooled to an amorphous solid. The single exception was a protein crystal where a nanocrystalline cryoprotectant had a protective effect. Lowering the pH, especially with ascorbic or nitric acids, had a protective effect, and SeMet lifetime increased monotonically with decreasing sample temperature (down to 93 K). The SeMet lifetime in one protein crystal was the same as that of the free amino acid, and the longest SeMet lifetime measured was found in the other protein crystal type. This protection was found to arise from the folded structure of the protein molecule. A mechanism to explain observed decay rates involving the damaging species following the electric field lines around protein molecules is proposed.

Radioprotectant screening for cryocrystallography

Southworth-Davies, R.J. — 2007-01-01

Radiation damage continues to present a problem to crystallographers using cryocooled protein crystals at third-generation synchrotrons. Free-radical scavengers have been suggested as a possible means of reducing the rate of this damage. The screening of a large number of potential radioprotectants was undertaken with an online microspectrophotometer using cystine and cysteine to model protein disulfide bonds and thiol groups, respectively. Oxidized α-lipoic acid was tested as a possible model disulfide bond. The evidence for the effectiveness of ascorbate as a radioprotectant was strengthened, and quinone, 2,2,6,6-tetramethyl-4-piperidone, and reduced dithiothreitol showed promise as radioprotectants.

X-ray radiation-induced damage in DNA monitored by online Raman

McGeehan, J.E. — 2007-01-01

High-quality Raman spectra have been collected on single DNA crystals kept at 100 K in a flow of cold nitrogen gas prior, during and after macromolecular crystallography (MX) data collection. The non-symmetric bending vibration for C—Br bonds in 8-bromo-2′-deoxyguanosine has been unambiguously identified at 293 cm−1 in the non-resonant Raman spectra. The breakage of this bond could be monitored by the continuous recording of Raman spectra during X-ray exposure, and its decay versus dose could be directly correlated with the loss of the bromide definition within the electron-density maps as determined with MX.

Plastic-embedded protein crystals

Ravelli, R.B.G. — 2007-01-01

Rapid vitrification followed by the replacement of the vitrified water by a solvent (freeze substitution) and then resin is a widely used procedure for preparing biological samples for electron microscopy. The resulting plastic-embedded samples permit convenient room-temperature sectioning (microtomy) and can yield well preserved cellular structures. Here this procedure has been applied to crystalline protein samples, and it is shown that it is possible to freeze-substitute vitrified crystals while preserving some of their original diffraction properties. The plastic-embedded crystals were used to collect a series of complete room-temperature data sets at a powerful macromolecular crystallography synchrotron beamline. Whereas one normally observes specific damage to disulfide bonds upon X-ray radiation, no such damage was seen for the plastic-embedded sample. The X-ray diffraction data allowed an initial atomic analysis to be made of the effects of freeze-substitution and plastic embedding on biological samples.

Current events

2006-11-01

Calibration of spectra from dispersive XAS beamlines

Ruffoni, M.P. — 2006-11-01

The DXAS Calibration computer program provides a quantitative and automated solution to the problem of calibrating spectra from dispersive XAS beamlines. Such spectra, obtained in arbitrary energy units, are calibrated with respect to the absorption features of a supplied reference spectrum, which has been obtained under similar conditions on a calibrated beamline. In addition to basic energy coordinate transformation parameters, DXAS Calibration supplies instrument corrections to compensate for mismatches in instrument response functions between the dispersive and reference beamlines.

Current events

2006-09-01

Stable top-up operation at SPring-8

Tanaka, H. — 2006-09-01

Top-up operation allows SPring-8 to provide highly stable X-ray beams with arbitrary filling patterns. The implementation of top-up operation is described, with a focus on the simultaneous achievement of stability of stored current, beam orbit, purity of an isolated single bunch, and beam injection efficiency. Stored-current fluctuations have been routinely reduced to a level of 10−3. Stored-beam oscillation on frequent beam injection, which was originally regarded as the most serious problem, has been successfully suppressed to a sufficiently low level that it never perturbs imaging experiments. Current impurities in nominally empty buckets have been reduced to a level of 10−9 over more than one week of operation, making possible the measurement of time-resolved spectra using high-current bunches. Finally, excellent injection efficiency, higher than 80%, is routinely obtained, even for small undulator gaps, which is critical for preventing radiation damage to insertion-device magnets and to reduce leakage radiation. The process of achieving highly stabilized top-up operation at SPring-8 and its utility for user experiments are described.

Current events

2006-07-01

Reciprocal space mapping and single-crystal scattering rods. Erratum

Smilgies, D.-M. — 2006-07-01

While assembling the high-resolution graphics for Fig. 2 of Smilgies et al. [J. Synchrotron Rad. (2005). 12, 807–811], the curve from the bottom panel was erroneously repeated in the top panel. Below is the corrected figure.

Current events

2006-05-01

Multiple-pass monochromators

Schoeffel, E. — 2006-05-01

Evaluation of imaging performance of a taper optics CCD `FReLoN' camera designed for medical imaging

Coan, P. — 2006-05-01

The purpose of this work was to assess the imaging performance of an indirect conversion detector (taper optics CCD `FReLoN' camera) in terms of the modulation transfer function (MTF), normalized noise power spectrum (NNPS) and detective quantum efficiency (DQE). Measurements were made with a synchrotron radiation laminar beam at various monochromatic energies in the 20–51.5 keV range for a gadolinium-based fluorescent screen varying in thickness; data acquisition and analysis were made by adapting to this beam geometry protocols used for conventional cone beams. The presampled MTFs of the systems were measured using an edge method. The NNPS of the systems were determined for a range of exposure levels by two-dimensional Fourier analysis of uniformly exposed radiographs. The DQEs were assessed from the measured MTF, NNPS, exposure and incoming number of photons. The MTF, for a given screen, was found to be almost energy independent and, for a given energy, higher for the thinnest screen. At 33 keV and for the 40 (100) µm screen, at 10% the MTF is 9.2 (8.6) line-pairs mm−1. The NNPS was found to be different in the two analyzed directions in relation to frequency. Highest DQE values were found for the combination 100 µm and 25 keV (0.5); it was still equal to 0.4 at 51.5 keV (above the gadolinium K-edge). The DQE is limited by the phosphor screen conversion yield and by the CCD efficiency. At the end of the manuscript the results of the FReLoN characterization and those from a selected number of detectors presented in the literature are compared.

Comments on A new model for statistical error analysis in XAS: about the distribution function of the absorption coefficient by E. Curis & S. Bénazeth (2001). J. Synchrotron Rad. 8, 264–266

Nadarajah, S. — 2006-05-01

Response to S. Nadarajah & S. Kotz's Comments on A new model for statistical error analysis in XAS: about the distribution function of the absorption coefficient by E. Curis & S. Bénazeth (2001). J. Synchrotron Rad. 8, 264–266

Curis, E. — 2006-05-01

Structure Determination by X-ray Crystallography. By Mark Ladd and Rex Palmer. Pp. xlii + 819. New York: Kluwer Academic/Plenum, 4th ed., 2003. Price (paperback) GBP 41, ISBN 0-306-47454-9.

Ohashi, Y. — 2006-05-01

Current events

2006-03-01

Introduction to the Special Issue on Detectors

Graafsma, H. — 2006-03-01

Current events

2006-01-01

Current events

2005-11-01

Deep UV and vacuum ultraviolet spectrophotometer

Schoeffel, E. — 2005-11-01

A transparent two-dimensional in situ beam-position and profile monitor for synchrotron X-ray beamlines

Kyele, N.R. — 2005-11-01

A compact, inexpensive and easy-to-construct two-dimensional in situ beam-position and profile monitor for synchrotron X-ray beamlines is presented. The device is based on the collection of spatially resolved scattered radiation from a polyimide foil. The X-ray beam passes through a foil placed in the path of the beam, which absorbs no more than 3% of the beam at 12 keV. The scattered radiation is collected at an angle of 90° through a collimator located below the foil onto a CCD sensor. The device was tested on bending-magnet beamline BM26 at the ESRF synchrotron radiation source and has a positional sensitivity better than 10 µm with a large working range of 25 mm × 25 mm. Although the device is optimized for use in the range 10–12 keV, it can easily be modified for use with higher-energy beams by using a suitably chosen scattering foil.

Synchrotron radiation and nanobiosciences – introductory overview

Pechkova, E. — 2005-11-01

Some introductory remarks on microbeam diffraction in nanobiosciences

Paris, O. — 2005-11-01

Current events

2005-09-01

1 m vacuum monochromator

Schoeffel, E. — 2005-09-01

Structure determination by single-crystal X-ray diffraction (SXD) at megabar pressures

Dera, P. — 2005-09-01

Structures from powders and poor-quality single crystals at high pressure

McMahon, M.I. — 2005-09-01

The use of single-crystal techniques and quasi-single-crystal samples in solving and refining complex crystal structures at high pressure is reviewed. In particular, recent studies of the incommensurate and modulated structures found in a number of elemental metals at high pressure are focused on.

Advances in array detectors for X-ray diffraction techniques

Hanley, Q.S. — 2005-09-01

Improved focal plane array detector systems are described which can provide improved readout speeds, random addressing and even be employed to simultaneously measure position, intensity and energy. This latter capability promises to rekindle interests in Laue techniques. Simulations of three varieties of foil mask spectrometer in both on- and off-axis configurations indicate that systems of stacked silicon detectors can provide energy measurements within 1% of the true value based on the use of single `foils' and ∼10000 photons. An eight-detector hybrid design can provide energy coverage from 4 to 60 keV. Energy resolution can be improved by increased integration time or higher flux experiments. An off-axis spectrometer design in which the angle between the incident beam and the detector system is 45° results in a shift in the optimum energy response of the spectrometer system. In the case of a 200 µm-thick silicon absorber, the energy optimum shifts from 8.7 keV to 10.3 keV as the angle of incidence goes from 0 to 45°. These new designs make better use of incident photons, lower the impact of source flicker through simultaneous rather than sequential collection of intensities, and improve the energy range relative to previously reported systems.

High-pressure polymorphism of cyclopentanol (C5H10O): the structure of cyclopentanol phase-V at 1.5 GPa

Moggach, S.A. — 2005-09-01

The fully ordered high-pressure crystal structure of cyclopentanol (C5H10O) has been solved using single-crystal X-ray diffraction techniques on station 9.8 at the SRS Daresbury Laboratory. At pressures above 1.5 GPa, cyclopentanol crystallizes in the monoclinic P21/c space group with a = 17.882 (3), b = 5.4573 (3), c = 9.6817 (14) Å, β = 104.699 (8)° and Z′ = 2. The crystal structure is characterized by the formation of hydrogen-bonded molecular chains, denoted C_2^2(4) in graph set notation, which lie parallel to the crystallographic c-axis, with the molecules adopting a pseudo fourfold arrangement around the central core of hydrogen bonds.

The effect of pressure on the crystal structure of hexagonal l-cystine

Moggach, S.A. — 2005-09-01

The crystal structure of hexagonal l-cystine has been determined at room temperature at pressures between 0.4 and 3.7 GPa; unit-cell dimensions were measured up to 6.4 GPa. The structure of this phase consists of molecules in their zwitterionic form, and crystallizes in the hexagonal space group P6122. The structure consists of hydrogen-bonded layers which are strongly reminiscent of those seen in α-glycine, and consist of R_4^4(16) hydrogen-bonded ring motifs. These layers are connected on one side by the disulfide bridges within the cystine molecules, and on the other by NH⋯O hydrogen bonds to other glycine-like layers. The most compressible unit-cell dimension, and the direction of greatest strain in the structure, is along the c-axis, and application of pressure pushes the layers closer together. The compression occurs approximately equally in the regions of the interlayer hydrogen bonds and the disulfide bridges; in the latter, changes in the C—S—S—C torsion angles allow the cystine molecules to act like springs. The effects of pressure can be interpreted in terms of closing-up of voids in the structure, and this leads to (i) a lessening of the N—C—C—O and C—S—S—C torsional angles, (ii) shortening of the N—H⋯O hydrogen bonds by 0.10–0.60 Å and (iii) a further shortening of an already short S⋯S contact from 3.444 (4) Å to 3.264 (4) Å.

Current events

2005-07-01

Variable-period undulators as synchrotron radiation sources. Erratum

Shenoy, G.K. — 2005-07-01

Citations are added to the paper by Shenoy et al. [J. Synchrotron Rad. (2003). 10, 205–213].

Application notes on the use of softer X-rays for anomalous powder diffraction

Cernik, R.J. — 2005-07-01

An in-vacuum diffractometer has been modified to collect powder diffraction data from a sample of promazine hydrochloride. Strong anomalous contrast has been observed at, or close to, the sulphur edge. Based on the data collected, a bespoke diffractometer has been designed that, together with minor changes to the geometry of the experiment, will enable the routine collection of powder data at absorption edges below 5.5 keV.

The uses of softer X-rays in structural studies

Cernik, R.J. — 2005-07-01

Nanoarchitectures of the animal extracellular matrix: opportunities for synchrotron radiation studies on collagen and fibrillin

Wess, T.J. — 2005-11-01

The extracellular matrix comprises structures that support the architectural organization of virtually all animal tissues. Within this architecture, two classes of protein assemblies found as long slender fibrils (collagen and fibrillin) characterize the bulk of the extracellular matrix. In both classes of fibrous protein, the molecular organization within a fibril ensures that the properties of the individual molecules transcend to the nanostructural and mesoscopic levels of structural organization and thence the tissue itself. The composition of the fibrils, in conjunction with other biomolecules and their suprafibrillar architecture, facilitates the formation of tissues as diverse as skin, tendon, cornea ciliary zonules and aorta. Here the relative tear resistance, strength, transparency and optical properties are paramount for proper function. Many structural investigations of fibrous protein structure have relied heavily on the use of synchrotron radiation in order to elucidate molecular packing, primarily due to the distinct benefits that X-ray diffraction provides, such as minimal sample preparation, rapid data collection and in situ mechanical testing. In this paper, an overview of the investigations that have revealed different levels of molecular architecture in fibril-based tissues is presented. Emerging future technology and how this can be matched with the pressing questions in extracellular matrix biology are also discussed.

Current events

2005-05-01

Resonant soft X-ray diffraction – in extremis

Hatton, P.D. — 2005-07-01

The use of softer-energy X-rays produced by synchrotron radiation for diffraction is an area of current interest. In this paper, experiments exploiting resonant scattering at the L absorption edges of 3d transition metal elements are reported. Such energies, typically 500–1000 eV, are at the extreme limit of soft X-ray diffraction where absorption effects are so severe that the sample and diffractometer must be placed in a windowless high-vacuum vessel. In addition, the Ewald sphere is so small as to likely contain, at most, only a single Bragg reflection. Advantages of using such radiation for the study of weak diffraction effects such as anomalous scattering, charge ordering, magnetic diffraction and orbital ordering are reported.

A high-throughput structural biology/proteomics beamline at the SRS on a new multipole wiggler

Cianci, M. — 2005-07-01

The North West Structural Genomics Centre's beamline, MAD10, at the SRS receives the central part of the radiation fan (0.5 mrad vertically, 4 mrad horizontally) produced by a new 2.46 T ten-pole wiggler. The optical arrangement of the beamline consists of a Rh-coated collimating Si mirror, a fixed-exit-beam double-crystal monochromator with sagittal bending for horizontal focusing and a second Rh-coated Si mirror for vertical focusing. The double-crystal Si (111) monochromator allows data collection in the 5–13.5 keV photon energy range with rapid (subsecond) tunability and high energy resolution. The monochromatic beam is optimized through a 200 µm collimator. The beamline end station has been designed around a Mar desktop beamline with high-throughput cryogenic sample changer, Mar225 CCD detector, liquid-N2 autofill system and an ORTEC C-TRAIN-04 energy-resolving high-count-rate X-ray fluorescence detector. The instrument is optimized for MAD/SAD applications in protein crystallography with the additional mode of operation of online single-crystal EXAFS studies on the same crystals. Thus, screening of metals/Se in the crystal can be performed quickly prior to MAD/SAD data collection by exciting the crystal with X-rays of appropriate energy and recording an energy-dispersive fluorescence spectrum. In addition, this experimental set-up allows for parallel XAFS measurements on the same crystal to monitor `radiation-induced' changes, if any, in e.g. the redox state of metal centres to be detected for a `metallic' functional group during crystallographic data collection. Moreover, careful minimization of the thickness of the Be window maximizes the intensity performance for the 2.0–2.5 Å softer wavelength range. This range also covers the K-edges of a number of important 3d transition metals as well as the L-edges of xenon and iodine and enhanced sulfur f ′′.

Overcoming protein denaturation caused by irradiation in a high-flux synchrotron radiation circular dichroism beamline

Janes, R.W. — 2005-07-01

It has been established that the new circular dichroism beamline CD12 has sufficiently high flux at low wavelengths to cause apparent irradiation problems with protein samples while their synchrotron radiation circular dichroism (SRCD) spectra are being collected. The cause of this effect has been extensively investigated and is reported in an accompanying paper [Wien et al. (2005). J. Synchrotron Rad. 12, 517–523.]. Experiments suggest that localized heating of the protein sample, leading to denaturation, is the probable cause. Methods to circumvent this problem by limiting the beam flux are reported. This was achieved using either an attenuation cell of water placed beam-side of the sample cell, or limiting the beam cross-sectional area hitting the sample. Such methods are shown to result in substantial reduction or apparent complete removal of this protein denaturation over the course of collecting three successive spectra. Elimination of this denaturation problem enables multiple SRCD scans for protein samples to be collected, which are vital both for good practice and for statistically valid results.

Towards an understanding of radiation damage in cryocooled macromolecular crystals

Nave, C. — 2005-05-01

Interest in radiation damage is growing rapidly owing to the surge in macromolecular crystallography experiments carried out at modern brilliant synchrotron macromolecular crystallography beamlines. Work on the characterization of radiation damage in cryocooled protein crystals is starting to have some impact on our understanding of the problem and of how damage might be affecting both the process of structure solution and the actual structure obtained. A brief review of the most recent developments is given together with an assessment of the remaining problems. Although progress is being made, the understanding of radiation damage is far from complete. Methods for recognizing the damage and treating the data are being made available but they are still at an early stage of development.

VUV irradiation effects on proteins in high-flux synchrotron radiation circular dichroism spectroscopy

Wien, F. — 2005-07-01

Synchrotron radiation circular dichroism (SRCD) spectroscopy is emerging as an important new tool in structural molecular biology. Previously we had shown that in lower-flux SRCD instruments, such as UV1 at ISA and beamline 3.1 at the SRS, vacuum ultraviolet (VUV) radiation damage to proteins was not evident after exposure over a period of hours. No effects were detected in either the protein primary or the secondary structures. However, with the development of high-flux beamlines, such as CD12 at the SRS, this issue has been revisited because of changes observed in the SRCD spectra of consecutive scans of protein samples obtained on this high-flux beamline. Experiments have been designed to distinguish between two different possible mechanisms: (i) photoionization causing free radicals or secondary electrons producing degradation of the protein, and (ii) local heating of the sample resulting in protein denaturation. The latter appears to be the principal source of the signal deterioration.

Brightness, spectral brightness or brilliance – Report of the Working Group on Synchrotron Radiation Nomenclature

Mills, D.M. — 2005-05-01

Current events

2005-03-01

Applications of synchrotron radiation to materials research

Shenoy, G. — 2005-03-01

Microfocus X-ray scattering investigations of eggshell nanotexture

Lammie, D. — 2005-11-01

The avian eggshell is a highly ordered calcitic bioceramic composite, with both inorganic and organic constituents. The interactions between the inorganic and organic components within the structure are poorly understood but are likely to occur at the nanometre level. Thus structural variation at this level may impinge on the overall structural integrity and mechanical performance of the eggshell, and therefore analysis at this level is fundamental in fully understanding this ordered structure. In this study, structural changes in the mineral crystallites were investigated by microfocus small-angle X-ray scattering (µSAXS) using synchrotron radiation. Small-angle X-ray scattering (SAXS) can be used to investigate structures on the nanometre scale such as size, shape, arrangement and internal porosity. A microfocused X-ray beam, 1.5 µm vertically by 7 µm, was used to produce vertical linear scans of the eggshell section. SAXS patterns were taken from the eggshell membrane (inner surface of the eggshell) to the cuticle (outer surface of the eggshell). This allowed textural variations within the eggshell to be mapped. The scattering intensity profile was then used to derive the dimension of scattering objects that define the nanotexture. The nanotexture observed may result from the presence of the organic matrix, which is embedded as intracrystalline particles producing voids within the calcified framework of large (>1 µm) calcite crystals. Porod analysis revealed the average size of a scattering interface to be approximately 4.5 nm with small changes that had a depth-dependent variation. These were largest at the mammillary layer/membrane boundary. The palisade layer displayed a small upward trend in size of scattering object. Parallel scans showed that the textural variations observed within the palisade layer are significant and indicate local subtextures. In addition, many of the patterns exhibit diffuse scattering streaks that could result from reflectivity from the larger crystallite interfaces. Changes in the orientation of diffuse streaks were observed within the different layers, the membranes, mammillary layer, palisade layer, vertical crystal layer and cuticle, indicating certain preferred orientations of the crystallites within the layers. The nanotextural variations that are apparent could have implications at the macroscopic level of the resulting eggshell.

Beam-size effects in radiation damage in insulin and thaumatin crystals

Schulze-Briese, C. — 2005-05-01

Cryocooled insulin and thaumatin crystals were irradiated in a series of alternating data collections and high-dose-rate exposures using either a vertically focused or vertically defocused beam. The main result is that the radiation damage is limited to the exposed region, which can be explained by the short range of the photoelectrons and the Auger electron cascade produced by light elements. Consequently, the unexposed angular range provides significantly improved data quality and electron density compared with previously exposed angular wedges of the crystal when a vertically focused beam is used, while no differences are observed between a fresh wedge and an exposed region for the vertically defocused beam. On the other hand, the focused beam provides higher I/σI ratios at high resolution than homogeneous sample illumination but also causes more rapid sample deterioration.

Parameters affecting the X-ray dose absorbed by macromolecular crystals

Murray, J.W. — 2005-05-01

The lifetime of a macromolecular crystal in an X-ray beam is assumed to be limited by the absorbed dose. This dose, expressed in Gray (Gy = J kg−1), is a function of a number of parameters: the absorption coefficients of the constituent atoms of the crystal, the number of molecules per asymmetric unit, the beam energy, flux, size and profile, the crystal size, and the total irradiation time. The effects of these variables on the predicted absorbed dose, calculated using the program RADDOSE, are discussed and are illustrated with reference to the irradiation of a selenomethionine protein crystal of unknown structure. The results of RADDOSE can and will in the future be used to inform the data collection procedure as it sets a theoretical upper limit on the total exposure time at a certain X-ray source. However, as illustrated with an example for which the experimental data are compared with prediction, the actual lifetime of a crystal could become shorter in those cases where specific damage breaks down crucial crystal contacts.

Phasing in the presence of radiation damage

Ravelli, R.B.G. — 2005-05-01

In the accurate estimation of small signals, redundancy of observations is often seen as an essential tool for the experimenter. This is particularly true during macromolecular structure determination by single-wavelength anomalous dispersion (SAD), where the exploitable signal can be less than a few percent. At the most intense undulator synchrotron beamlines, the effect of radiation damage can be such that all usable signal is obscured. Here the magnitude of this effect in experiments performed at the Se K-edge is quantified. Six successive data sets were collected on the same crystal, interspersed with two exposures to the X-ray beam during which data were not collected. It is shown that the very first data set has excellent phasing statistics, whereas these statistics degrade for the later data sets. Merging several data sets into one, highly redundant, data set only gave moderate improvements as a result of the presence of radiation damage. Part of the damage could be corrected for using a linear interpolation scheme. Interpolation of the data to a low-dose as well as to a high-dose data set allowed us to combine the SAD method with the radiation-damage induced phasing (RIP) technique, which further improved the experimental phases, especially after density modification. Some recommendations are given on how to mitigate the effect of radiation damage during structure determination.

Current events

2005-01-01

Metallogenomics and biological X-ray absorption spectroscopy

Ascone, I. — 2005-01-01

An overview of the second special issue of the journal on biological applications of X-ray absorption spectroscopy (BioXAS) is presented. The emphasis is on the study of metalloproteins in the context of structural genomics programmes (metallogenomics).

The use of softer X-rays in the structure elucidation of microporous materials

Helliwell, M. — 2005-07-01

Microporous materials, such as zeolites and aluminophosphates, have many applications as molecular sieves and shape-selective catalysts. This is due to their three-dimensional frameworks, which contain regular pores and channels, to their high acidity, arising from Brønsted and Lewis acid active sites, and to the incorporation of transition metal atoms into framework sites. This review firstly provides an introduction into the nature and properties of these materials, and their important applications; the difficulties in their full characterization and possible methods of elucidating their structures are then outlined; finally, methods of characterization, utilizing `softer X-rays' are introduced. The first method is the determination of low concentrations of transition metals, incorporated into the frameworks using single crystal anomalous dispersion crystallography; synchrotron radiation is used to tune to the absorption edge of the metal atom in question, in order to change its signal relative to that of the rest of the structure, thereby allowing the pinpointing of its positions and the determination of its concentration at each site in the framework. Secondly, the use of longer wavelengths in powder diffraction studies is described, which, by stretching out the powder pattern, thereby reduces the overlapping of the diffraction peaks, thus allowing the structure to be solved by conventional direct methods. Finally, the use of X-ray absorption spectroscopy to determine the metal incorporation and the nature of coordination at the metal atom sites, in Mn silicalite-1 and FAPO-36, are described.

An internet-based synchrotron experiment for students measuring the X-ray magnetic circular dichroism of a PtFe alloy

Paulus, M. — 2005-03-01

A new internet-based synchrotron experiment for students is presented. A polarimeter and computer software have been developed for measuring via the internet the X-ray magnetic circular dichroism of PtFe around its Pt LII and LIII absorption edges. From the experiment, students can examine the X-ray magnetic circular dichroism of a thin PtFe foil utilizing circular-polarized synchrotron radiation emitted by the superconducting asymmetric wiggler at the synchrotron radiation source DELTA of the University of Dortmund.

Specimen charging in X-ray absorption spectroscopy: correction of total electron yield data from stabilized zirconia in the energy range 250–915 eV

Vlachos, D. — 2005-03-01

The effects of specimen charging on X-ray absorption spectroscopy using total electron yield have been investigated using powder samples of zirconia stabilized by a range of oxides. The stabilized zirconia powder was mixed with graphite to minimize the charging but significant modifications of the intensities of features in the X-ray absorption near-edge fine structure (XANES) still occurred. The time dependence of the charging was measured experimentally using a time scan, and an algorithm was developed to use this measured time dependence to correct the effects of the charging. The algorithm assumes that the system approaches the equilibrium state by an exponential decay. The corrected XANES show improved agreement with the electron energy-loss near-edge fine structure obtained from the same samples.

Towards the high-throughput expression of metalloproteins from the Mycobacterium tuberculosis genome

Hall, J.F. — 2005-01-01

The provision of high-quality protein in adequate quantities is a prerequisite for any structural genomics programme. A number of proteins from the Mycobacterium tuberculosis genome have been expressed and the success at each stage of the process assessed. Major difficulties have been encountered in the purification and solubilization of many of these proteins, most likely as a result of mis-folding. Some improvements have been made to the protocol but the overall success rate is still limited; however, the use of a cell-free protein expression system will circumvent some of the difficulties encountered. Alternative purification systems are also required and the properties of a mutant blue copper protein are described, that may offer a combined purification and tagging system.

Heterologous metalloprotein biosynthesis in Escherichia coli: conditions for the overproduction of functional copper-containing nitrite reductase and azurin from Alcaligenes xylosoxidans

Harris, R.L. — 2005-01-01

This paper reports on the optimization of conditions for the overproduction and isolation of two recombinant copper metalloproteins, originally encoded on the chromosome of the dentrifying soil bacterium Alcaligenes xylosoxidans, in the heterologous host Escherichia coli. The trimeric enzyme nitrite reductase (NiR) contains both type-1 and type-2 Cu centres, whilst its putative redox partner, azurin I, is monomeric and has only a type-1 Cu centre. Both proteins were processed and exported to the periplasm of E. coli, which is consistent with their periplasmic location in their native host A. xylosoxidans. NiR could be readily purified from the periplasmic fraction of E. coli but the enzyme as isolated possessed only type-1 Cu centres. The type-2 Cu centre could be fully reconstituted by incubation of the periplasmic fraction with copper sulfate prior to enzyme purification. Azurin I could only be isolated with a fully occupied type-1 centre when isolated from the crude cell extract but not after isolation from the periplasmic fraction, suggesting loss of the copper due to proteolysis. Based on a number of criteria, including spectroscopic, mass spectrometric, biochemical and structural analyses, both recombinant proteins were found to be indistinguishable from their native counterparts isolated from A. xylosoxidans. The findings of this work have important implications for the overproduction of recombinant metalloproteins in heterologous hosts.

Softer and soft X-rays in macromolecular crystallography

Djinović Carugo, K. — 2005-07-01

The utilization and the potential of softer and soft X-rays in macromolecular crystallography as well as the challenges associated with the corresponding diffraction experiments and their possible remedies are reviewed.

Current events

2004-11-01

Comments on Treatment of EXAFS data taken in the fluorescence mode in non-linear conditions by G. Ciatto et al. (2004). J. Synchrotron Rad. 11, 278–283

Warburton, W.K. — 2004-11-01

Response to W. K. Warburton's Comments on Treatment of EXAFS data taken in the fluorescence mode in non-linear conditions by G. Ciatto et al. (2004). J. Synchrotron Rad. 11, 278–283

Ciatto, G. — 2004-11-01

Aberration corrected vacuum ultraviolet monochromator

Schoeffel, E. — 2004-09-01

The BioCAT undulator beamline 18ID: a facility for biological non-crystalline diffraction and X-ray absorption spectroscopy at the Advanced Photon Source

Fischetti, R. — 2004-09-01

The 18ID undulator beamline of the Biophysics Collaborative Access Team at the Advanced Photon Source, Argonne, IL, USA, is a high-performance instrument designed for, and dedicated to, the study of partially ordered and disordered biological materials using the techniques of small-angle X-ray scattering, fiber diffraction, and X-ray absorption spectroscopy. The beamline and associated instrumentation are described in detail and examples of the representative experimental results are presented.

Carl-Ivar Brändén, 1934–2004

Schneider, G. — 2004-09-01

Cullie Sparks, 1929–2004

Ice, G.E. — 2004-07-01

Image quality improvement in a hard X-ray projection microscope using total reflection mirror optics

Mimura, H. — 2004-07-01

A new figure correction method has been applied in order to fabricate an elliptical mirror to realize a one-dimensionally diverging X-ray beam having high image quality. Mutual relations between figure errors and intensity uniformities of diverging X-ray beams have also been investigated using a wave-optical simulator and indicate that figure errors in relatively short spatial wavelength ranges lead to high-contrast interference fringes. By using a microstitching interferometer and elastic emission machining, figure correction of an elliptical mirror with a lateral resolution close to 0.1 mm was carried out. A one-dimensional diverging X-ray obtained using the fabricated mirror was observed at SPring-8 and evaluated to have a sufficiently flat intensity distribution.

A large-area CMOS imager as an X-ray detector for synchrotron radiation experiments

Yagi, N. — 2004-07-01

The performance of a CMOS flatpanel imager from Hamamatsu Photonics (C7942) has been tested in various synchrotron radiation experiments. This detector has a detection area of about 120 mm × 120 mm with 0.05 mm pixels, and a frame rate of 2 s−1. The commercially available product was insensitive to X-rays with an energy lower than 15 keV, but slight modifications solved this problem. Images obtained in small-angle scattering, protein crystallography and medical imaging experiments were all of high quality. The fast readout and the large area are advantageous in real-time imaging. Although its noise level is higher than the area detectors that are currently used in synchrotron radiation experiments, it is particularly useful in experiments where other bulky detectors cannot be used. Its relatively low price (about USD 30000) makes it a unique option in the choice of detectors.

Soft X-ray resonant magnetic scattering from a Ni layer with modulated magnetic anisotropy

Haznar, A. — 2004-05-01

Soft X-ray resonant magnetic scattering studies on a magnetically modulated, but nominally chemically homogenous, 5 nm Ni layer in a Cu/Ni/Cu/Co/NiO/GaAs(110) system are reported. It was possible to estimate the main chemical structure of the sample on the basis of the results from specular reflectivity and rocking scans, probing the scattering vector components in the perpendicular and in-plane direction to the Ni wires, respectively. The magnetic scattering using polarized X-rays demonstrates the magnetic modulation of the Ni layer.

XAFS imaging of Tsukuba gabbroic rocks: area analysis of chemical composition and local structure

Mizusawa, M. — 2004-03-01

Gabbroic rocks were collected at Mount Tsukuba in Japan, and their XAFS images were studied using a projection-type X-ray fluorescence (XRF) microscope, which is a powerful new tool recently developed for extremely rapid imaging. The instrument employs a grazing-incidence arrangement in order that primary X-rays illuminate the whole sample surface, as well as parallel-beam optics and an extremely close geometry in order to detect XRF by a high-performance X-ray CCD system with 1024 × 1024 pixels. The XRF image indicated that black amphibole and white feldspar, both of which are typical mineral textures of the rock, contain iron. The origin has been suggested to be several small yellowish-brown minerals contained there. The XAFS imaging has been carried out by repeating the exposure of XRF images during the energy scan of the primary X-rays. It has been found that the structure is qualitatively close to that of olivine, and the main differences found in both areas can be explained as a difference in iron and magnesium concentration, i.e. the mixed ratio of forsterite (Mg2SiO4) and fayalite (Fe2SiO4). The feasibility of the present XAFS imaging method has been demonstrated for realistic inhomogeneous minerals.

SOLVE and RESOLVE: automated structure solution, density modification and model building

Terwilliger, T. — 2004-01-01

The software SOLVE and RESOLVE can carry out all the steps in macromolecular structure solution, from scaling and heavy-atom location through phasing, density modification and model-building in the MAD, SAD and MIR cases. SOLVE uses scoring scheme to convert the decision-making in macromolecular structure solution to an optimization problem. RESOLVE carries out the identification of NCS, density modification and automated model-building. The procedure is fully automated and can function at resolutions as low as 3 Å.

Recent developments in the PHENIX software for automated crystallographic structure determination

Adams, P.D. — 2004-01-01

A new software system called PHENIX (Python-based Hierarchical ENvironment for Integrated Xtallography) is being developed for the automation of crystallographic structure solution. This will provide the necessary algorithms to proceed from reduced intensity data to a refined molecular model, and facilitate structure solution for both the novice and expert crystallographer. Here, the features of PHENIXare reviewed and the recent advances in infrastructure and algorithms are briefly described.

Third-Generation Hard X-ray Synchrotron Radiation Sources. Edited by D. M. Mills. New York: John Wiley, 2002. Price GBP 69.95, ISBN 0-471-31433-1.

Schneider, J. — 2003-09-01

XAFS studies of nitrogenase: the MoFe and VFe proteins and the use of crystallographic coordinates in three-dimensional EXAFS data analysis. Erratum

Strange, R.W. — 2003-03-01

One of the authors was omitted in the published version of the paper by Strange et al. [J. Synchrotron Rad. (2003), 10, 71–75]. The full author list is given above. Since the acceptance of our paper, an atomic-resolution (1.16 Å) structure of MoFe-protein has emerged [Einsle et al. (2002), Science, 297, 1696–1700]. We take this opportunity to update Table 1 of the paper, demonstrating improved agreement of the three-dimensional XAFS refinement with atomic-resolution metrical information.

Multiple-electron excitation in X-ray absorption: a simple generic model. Erratum

Roy, M. — 2003-03-01

Two typographical errors have been observed in the paper by Roy et al. [J. Synchrotron Rad. (2001), 8, 1103–1108]. Equation (16) should read\eqalign{P(\hbar\omega)={}&P(\infty)\left[{{{\pi(2n-1)!}\over{2^{2n}(n-1)!(n+1)!}}}\right]^{-1}E_B^{\,n+1/2}\cr&\times\int\limits_0^{E_{\max}}{{{E^{1/2}}\over{(E_B+E)^{n+2}[1+t_{\,o}^{\,2}\,(E_B+E)^2]}}}\,\,{\rm{d}}E,}while the correct form of equation (18) is\eqalign{P(\hbar\omega)={}&P(\infty)\left[{{{\pi(2n-1)!}\over{2^{2n}(n-1)!(n+1)!}}}\right]^{-1}\cr&\times\int\limits_1^{E_p/E_B}{{{(X-1)^{1/2}}\over{X^{n+2}[1+{\textstyle{1\over4}}\left({n-1}\right)^2(E_B/E_p)X^2]}}}\,\,{\rm{d}}X.}These typographical errors do not affect our results or conclusions.

Comments on Synchrotron fibre diffraction identifies and locates foetal collagenous breast tissue associated with breast carcinoma by V. J. James (2002). J. Synchrotron Rad. 9, 71–76

Suortti, P. — 2003-03-01

Comments on Synchrotron fibre diffraction identifies and locates foetal collagenous breast tissue associated with breast carcinoma by V. J. James (2002). J. Synchrotron Rad. 9, 71–76

Rogers, K.D. — 2003-03-01

Response to P. Suortti et al.'s and K. D. Rogers et al.'s Comments on Synchrotron fibre diffraction identifies and locates foetal collagenous breast tissue associated with breast carcinoma by V. J. James (2002). J. Synchrotron Rad. 9, 71–76

James, V. — 2003-03-01