Anomalous X-ray diffraction with soft X-ray synchrotron radiation.

Anomalous diffraction with soft X-ray synchrotron radiation opens new possibilities in protein crystallography and materials science. Low-Z elements like silicon, phosphorus, sulfur and chlorine become accessible as new labels in structural studies. Some of the heavy elements like uranium exhibit an unusually strong dispersion at their M(V) absorption edge (lambdaMV = 3.497 A, E(MV) = 3545 eV) and so does thorium. Two different test experiments are reported here showing the feasibility of anomalous X-ray diffraction at long wavelengths with a protein containing uranium and with a salt containing chlorine atoms. With 110 electrons the anomalous scattering amplitude of uranium exceeds by a factor of 4 the resonance scattering of other strong anomalous scatterers like that of the lanthanides at their L(III) edge. The resulting exceptional phasing power of uranium is most attractive in protein crystallography using the multi-wavelength anomalous diffraction (MAD) method. The anomalous dispersion of an uranium derivative of asparaginyl-tRNA synthetase (hexagonal unit cell; a = 123.4 A, c = 124.4 A) has been measured for the first time at 4 wavelengths near the M(V) edge using the beamline ID1 of ESRF (Grenoble, France). The present set up allowed to measure only 30% of the possible reflections at a resolution of 4 A, mainly because of the low sensitivity of the CCD detector. In the second experiment, the dispersion of the intensity of 5 X-ray diffraction peaks from pentakismethylammonium undecachlorodibismuthate (PMACB, orthorhombic unit cell; a = 13.003 A, b = 14.038 A, c = 15.450 A) has been measured at 30 wavelengths near the K absorption edge of chlorine (lambdaK = 4.397 A, EK= 2819.6 eV). All reflections within the resolution range from 6.4 A to 3.4 A expected in the 20 degree scan were observed. The chemical state varies between different chlorine atoms of PMACB, and so does the dispersion of different Bragg peaks near the K-edge of chlorine. The results reflect the performance of the beamline ID1 of ESRF at wavelengths beyond 3 A at the end of 1998. A gain by a factor 100 for diffraction experiments with 4.4 A photons was achieved in Autumn 1999 when two focusing mirrors had been added to the X-ray optics. Further progress is expected from area detectors more sensitive to soft X-rays. Both CCD detectors and image plates would provide a gain of two orders of measured intensity. Image plates would have the additional advantage that they can be bent cylindrically and thus cover a larger solid angle in reciprocal space. In many cases, samples need to be cooled: closed and open systems are presented. A comparison with the state of art of soft X-ray diffraction, as it had been reached at HASYLAB (Hamburg, Germany), and as it is developing at the Brookhaven National Laboratory (USA), is given.     

Determination of trace elements in Parkinson's diseased brain tissue using microbeam of synchrotron radiation

The microbeam synchrotron X-ray fluorescence (MSXRF) technique is powerful tool for determining elemental composition and spatial distribution of elements within materials on the microscopic level. Therefore, application of this technique seems to be helpful for investigation of the role of trace elements in neurodegenerative processes. As a preliminary study the MSXRF technique was applied for elemental microanalysis in cryo-sections of substantia nigra tissue for cases of Parkinson's disease (PD) and the control. Two-dimensional maps of elemental distribution showed significant topographic and quantitative differences between the pathological and control tissues. Quite interesting is significant increased accumulation of Ca, Cu, Fe, Fe, and Zn that was observed in PD neuron perikaryal parts.     

Efficient use of synchrotron radiation for macromolecular diffraction data collection

In recent years, number of X-ray synchrotron beam lines dedicated to collecting diffraction data from macromolecular crystals has exceeded 50. Indeed, today most protein and nucleic acid crystal structures are solved and refined based on the synchrotron data. Collecting diffraction data on a synchrotron beam line involves many technical points, but it is not a mere technicality. Even though the available hardware and software have become more advanced and user-friendly, it is always beneficial if the experimenter is aware of the problems involved in the data collection process and can make informed decisions leading to the highest possible quality of the acquired diffraction data. Various factors, important for the success of data collection experiments and their relevance for different kinds of applications, are discussed.     

A synchrotron radiation X-ray diffraction study of the crystallinity of tendon fibres

The equatorial diffraction pattern of tendon collagen fibres was measured during short successive exposures at different lengths using a double focusing X-ray synchrotron radiation camera with film and with an area detector. Similarly, patterns from thin fibres from premature rats were recorded. The patterns unambiguously illustrate the relationship between fibre crystallinity and the age of the animal. Further, the results indicate that in the initial part of the linear region of the stiffness-versus-length curve, the collagen fibres are characterized by a quasihexagonal arrangement of collagen molecules, whereas at the end of this region, the molecular arrangement becomes hexagonal.     

Energy dispersive X-ray diffraction by collagen fibrils in costal cartilage using synchrotron radiation

Low-angle X-ray diffraction patterns from the isotropic distribution of collagen fibrils, which occur in low concentrations in costal cartilage, were recorded using synchrotron radiation. An energy dispersive technique was used to exploit the properties of synchrotron radiation to the full. The third, fourth, fifth and sixth diffraction orders from the axial periodicity of the fibrils were recorded and used to calculate a value for this periodicity of 67 ± 1 nm. This result is in good agreement with measurements made on amianthoid areas as well as from fibrils in tendon, which consist of a chemically distinct form of collagen.     

Optimizing dose enhancement with Ta2O5 nanoparticles for synchrotron microbeam activated radiation therapy

Microbeam Radiation Therapy (MRT) exploits tumour selectivity and normal tissue sparing with spatially fractionated kilovoltage X-ray microbeams through the dose volume effect. Experimental measurements with Ta₂O₅ nanoparticles (NPs) in 9L gliosarcoma treated with MRT at the Australian Synchrotron, increased the treatment efficiency. Ta₂O₅ NPs were observed to form shells around cell nuclei which may be the reason for their efficiency in MRT. In this article, our experimental observation of NP shell formation is the basis of a Geant4 radiation transport study to characterise dose enhancement by Ta₂O₅ NPs in MRT. Our study showed that NP shells enhance the physical dose depending microbeam energy and their location relative to a single microbeam. For monochromatic microbeam energies below ∼70 keV, NP shells show highly localised dose enhancement due to the short range of associated secondary electrons. Low microbeam energies indicate better targeted treatment by allowing higher microbeam doses to be administered to tumours and better exploit the spatial fractionation related selectivity observed with MRT. For microbeam energies above ∼100 keV, NP shells extend the physical dose enhancement due to longer-range secondary electrons. Again, with NPs selectively internalised, the local effectiveness of MRT is expected to increase in the tumour. Dose enhancement produced by the shell aggregate varied more significantly in the cell population, depending on its location, when compared to a homogeneous NP distribution. These combined simulation and experimental data provide first evidence for optimising MRT through the incorporation of newly observed Ta₂O₅ NP distributions within 9L cancer cells.     

Time-resolved X-ray diffraction from tendon collagen during creep using synchrotron radiation

In order to understand the molecular mechanism of relaxation phenomena in collagenous tissue, time-resolved, small-angle X-ray diffraction measurements were performed on bovine Achilles tendon collagen under creep. A tension-induced increase in the 67 nm period (D-period) was observed, and the strain in the D-period, epsilon D, was found to be almost proportional to the external force per unit cross-sectional area (average stress) of the specimen. With an increase in epsilon D, a change in the ratio of intensities of the third-order reflection peak of the D-period to that of the second-order peak was also observed. The increase in epsilon D was decomposed into three elementary processes of D-period deformation, which are presented on the basis of the Hodge-Petruska model: (1) molecular elongation, (2) increase in gap region, and (3) relative slippage of lateral adjoining molecules. Up to 8 MPa of average stress, the contribution to epsilon D originated mostly from only mode (1). At more than 10 MPa of average stress, modes (2) and (3) also contributed to fibril elongation. For epsilon D by molecular elongation (mode (1)), the time dependence of the D-period change in the immediate response region is a sharply shaped step function, while the contribution to epsilon D by molecular rearranging modes gives a slight creep nature at the immediate response region in the time dependence of epsilon D. Because this creep nature is observed at the immediate response, it is related qualitatively to the KWW function in a stress-relaxation modulus of collagenous tissue observed in an immediate response region (Sasaki et al. (1993). Journal of Biomechanics 26, 1369-1376). The elementary process of KWW-type relaxation is concluded to be related to the tension-induced molecular rearrangement within a D-period.     

Position of caesium ions in crystalline B-form DNA determined by synchrotron radiation diffraction. Preliminary results

The diffraction patterns of CsDNA taken with copper X-irradiation are considerably impaired because of the strong X-ray absorption by caesium ions. The use of high power synchrotron radiation of wavelength λ = 1.2 Å has yielded photographs suitable for intensity measurements.The structure of phage T2 CsDNA at 76% relative humidity is isomorphous to the crystalline B-form of LiDNA, and the disposition of cations appears to conform to the 10-fold screw symmetry of B-DNA. The structure factor amplitudes of 20 reflections in the diffraction pattern of phage T2 CsDNA are noticeably different from those of the same reflections in the LiDNA diffraction pattern, and the positions of the Cs⁺ ions could thus be found. The best model has the cations located significantly close to dyad axes lying between the planes of successive nucleotide pairs. One of the two cations “belonging” to a nucleotide pair touches the surface of the narrow groove of the B-DNA double helix, while the other is on the wide groove side, rather far from its “own” DNA molecule.     

The tertiary structure of azurin from Pseudomonas denitrificans as determined by Cu resonant diffraction using synchrotron radiation

The structure of Pseudomonas denitrificans azurin has been solved at 3.0 A resolution by film data collection methods using synchrotron radiation. Bijvoet pairs of reflections were collected using 8.98 keV radiation where both delta f' and delta f", the real and imaginary corrections to azurin's Cu prosthetic group scattering, respectively, are large, and using 8.00 keV radiation where these corrections are smaller. The Cu atom was located by difference Patterson syntheses and used to phase the observed protein structure-factor moduli of selected reflections. An atomic model of the protein was built using a restricted data set and phases for all observed reflections to 3.0 A resolution were subsequently calculated from the initial model. The atomic model was subsequently rebuilt using all observed data. The results of this work are presented here and illustrate the utility of synchrotron radiation phasing techniques in solving the structures of metalloproteins without recourse to multiple isomorphous replacement.     

Time-resolved X-ray diffraction studies of frog skeletal muscle isometrically twitched by two successive stimuli using synchrotron radiation

In order to clarify the delay between muscular structural changes and mechanical responses, the intensity changes of the equatorial and myosin layer-line reflections were studied by a time-resolved X-ray diffraction technique using synchrotron radiation. The muscle was stimulated at 12-13 degrees C by two successive stimuli at an interval (80-100 ms) during which the second twitch started while tension was still being exerted by the muscle. At the first twitch, the intensity changes of the 1.0 and 1.1 equatorial reflections reached 65 and 200% of the resting values, and further changes to 55 and 220% were seen at the second twitch, respectively. Although the second twitch decreased not only the time to peak tension but also that to the maximum intensity changes of the equatorial reflections (in both cases, about 15 ms), the delay (about 20 ms) between the intensity changes and the development of tension at the first twitch were still observed at the second twitch. On the other hand, the intensities of the 42.9 nm off-meridional and the 21.5 nm meridional myosin reflections decreased at the first twitch to the levels found when a muscle was isometrically tetanized, and no further decrease in their intensities was observed at the second twitch. These results indicate that a certain period of time is necessary for myosin heads to contribute to tension development after their arrival in the vicinity of the thin filaments during contraction.     

In situ biological dose mapping estimates the radiation burden delivered to 'spared' tissue between synchrotron X-ray microbeam radiotherapy tracks

Microbeam radiation therapy (MRT) using high doses of synchrotron X-rays can destroy tumours in animal models whilst causing little damage to normal tissues. Determining the spatial distribution of radiation doses delivered during MRT at a microscopic scale is a major challenge. Film and semiconductor dosimetry as well as Monte Carlo methods struggle to provide accurate estimates of dose profiles and peak-to-valley dose ratios at the position of the targeted and traversed tissues whose biological responses determine treatment outcome. The purpose of this study was to utilise γ-H2AX immunostaining as a biodosimetric tool that enables in situ biological dose mapping within an irradiated tissue to provide direct biological evidence for the scale of the radiation burden to 'spared' tissue regions between MRT tracks. Γ-H2AX analysis allowed microbeams to be traced and DNA damage foci to be quantified in valleys between beams following MRT treatment of fibroblast cultures and murine skin where foci yields per unit dose were approximately five-fold lower than in fibroblast cultures. Foci levels in cells located in valleys were compared with calibration curves using known broadbeam synchrotron X-ray doses to generate spatial dose profiles and calculate peak-to-valley dose ratios of 30-40 for cell cultures and approximately 60 for murine skin, consistent with the range obtained with conventional dosimetry methods. This biological dose mapping approach could find several applications both in optimising MRT or other radiotherapeutic treatments and in estimating localised doses following accidental radiation exposure using skin punch biopsies.     

A microbeam X-ray diffraction study of insulin spherulites

The peptide hormone insulin forms a spherical aggregate, called a spherulite, at low pH and high temperature. A spherulite is composed of a core and many fibrils extending from it. These fibrils are thought to be amyloid fibers with a beta-sheet structure. In the present study, spherulites with a diameter of 50-100 microm were examined by X-ray fiber diffraction using a 6 microm beam. When a spherulite was scanned with the microbeam and the observed diffraction patterns were arranged in a two-dimensional array, the direction of the scatter was centrosymmetric, demonstrating a symmetric growth of fibrils. There were diffraction peaks at Bragg spacings of 23 nm, 3.3 nm and 1.2 nm in the direction perpendicular to the fibrils and 0.48 nm along the fibrils. The 0.48 nm reflection shows that the hydrogen bonds between beta-strands are along the fibril. The 23 nm reflection corresponds to the separation between fibrils, the 3.3 nm reflection is due to the arrangement of protofilaments, and the 1.2 nm reflection arises from the arrangement of peptide chains. On the basis of these results, a model of a fibril with an extended insulin molecule is proposed.     

Microradiosurgical cortical transections generated by synchrotron radiation

PurposeMicroplanar X-ray beams (microbeams) originated by synchrotron sources have been delivered to the visual brain cortex regions in rodents to create microscopically narrow lesions. The effects of microbeams mimic those generated by microsurgical subpial transections (also known as multiple subpial transections) but are obtained in a low-invasive way.MethodsImage-guided atlas-based microbeam cortical transections have been generated on seven 1 month-old Wistar rats. An array of 10 parallel beams of 25 microns in thickness and spaced of 200 micron center-to-center was centered on the visual cortex and deposited an incident dose of 600 Gy.ResultsThe procedure was well tolerated by rats. After recovery, rats showed regular behavior, no sign of gross visual impairment and regular weight gain. After 3 months, rats were sacrificed and brains histologically examined. Cortical transections resembling those obtained through a surgical incision were found over the irradiated region. Remarkable sparing of the cortical columns adjacent to the transections was observed. No sign of radionecrosis was evident at least at this time point.ConclusionsThe visual brain cortex transected by synchrotron-generated microbeams showed an incision-like path of neuronal loss while adjacent non irradiated columns remained intact. These preliminary findings, to be further investigated also using other techniques, suggest that microbeam radiosurgery can affect the cortex at a cellular level providing a potential novel and attractive tool to study cortical function.     

X-ray diffraction evidence of collagen molecular packing and cross-linking in fibrils of rat tendon observed by synchrotron radiation.

X-ray diffraction patterns of fibres from 90 day (mature) rat-tail tendons were investigated using synchrotron radiation. The specimens were kept isometric at their corresponding in vitro rest length, and effects of pH and ionic strength were studied during short X-ray exposures. The results indicate that fibrils, equilibrated in physiological Ringer prior to exposure, have segregated lateral regions of well ordered collagen molecular packing. Lowering the ionic strength or the pH to 4.0 causes an order/disorder transition during which the fibril crystallinity decreases. At pH 3.5 a dramatic increase in the lateral swelling was observed. This effect was absent for fibres pretreated with sodium borohydride. The results are interpreted on the basis of cross-linking phenomena whereby the aldimine cross-link seems to be a controlling component of the lateral packing arrangement of collagen molecules.     

Nuclear forward scattering of synchrotron radiation by deoxymyoglobin

Nuclear forward scattering of synchrotron radiation is used to determine the quadrupole splitting and the mean square displacement of the iron atom in deoxymyoglobin in the temperature range between 50 K and 243 K. Above 200 K an abnormally fast decay of the forward scattered intensity at short times after the synchrotron flash is observed, which is caused by protein-specific motions. The results strongly support the picture that protein dynamics seen at the position of the iron can be understood by harmonic motions in the low temperature regime while in the physiological regime diffusive motions in limited space are present. The shape of the resonance broadening function is investigated. An inhomogeneous broadening with a Lorentzian distribution indicating dipole interactions results in a better agreement with the experimental data than the common Gaussian distribution. Received: 30 August 1999 / Revised version: 22 October 1999 / Accepted: 6 December 1999     

Chemical and physical properties of sulfated silk fabrics

Silk fabrics were treated with chlorosulphonic acid in pyridine for different times. The amount of sulfur bound to silk increased during the first 2 h of reaction and then reached a plateau. The amino acidic pattern of sulfated silk remained essentially unchanged for short reaction times (< or =2 h). Longer reaction times resulted in drastic changes in the concentration of Asp, Glu, and Tyr. Surface morphology and texture of silk fabrics changed upon sulfation. Warp and weft yarns became progressively thinner, and deposits of foreign material appeared on the fiber surface. Changes were more evident at longer reaction times (> or =2 h). Spectroscopic analyses performed by FT-IR and FT-Raman showed the appearance of new bands attributable to various vibrations of sulfated groups. The IR bands at 1049 and 1014 cm-1, due to organic sulfate salts, were particularly intense. Bands assigned to alkyl sulfates and sulfonamides appeared in the 1300-1180 cm-1 range. Organic covalent sulfates displayed a weak but distinct IR band at 1385 cm-1. Both IR and Raman spectra revealed that silk fibroin mainly bound sulfates through the hydroxyl groups of Ser and Tyr, while involvement of amines could not be proved. Changes observed in the amide I and II range indicated an increase of the degree of molecular disorder of sulfated silk. Accordingly, the I850/I830 intensity ratio between the two Tyr bands at 850-830 cm-1 increased from 1.41 to 1.52, indicating a more exposed state of Tyr residues in sulfated silk. TGA, DSC, and TG analyses showed that sulfated silk attained a higher thermal stability. A thermal transition attributable to sulfated silk fibroin fractions appeared at about 260 degrees C in the DSC thermograms.     

X-ray diffraction photographs of chicken gizzard G-actin.DNase I complex crystals taken with synchrotron radiation

X-ray diffraction photographs of a chicken gizzard G-actin.DNase I complex crystal have been recorded using the synchrotron radiation beam emitted by the Synchrotron Radiation Source at Daresbury and the Photon Factory at Tsukuba. The resolution limit was extended to 2.4 A and the exposure time was reduced approximately by a factor of 10, when data recorded at the Photon Factory, were compared with those recorded with a conventional rotating-anode source. Using a newly designed Weissenberg camera equipped with a multi-layer line screen, the diffraction data in a 36 degrees oscillation range were recorded on a single film up to 3.5 A resolution.     

Sulfur distribution in bacteriorhodopsin from multiple wavelength anomalous diffraction near the sulfur K-edge with synchrotron x-ray radiation.

Bacteriorhodopsin contains nine sulfur atoms from the nine methionine residues. The distribution of these sulfur atoms in the projected density map was determined from x-ray diffraction experiments using multiple wavelength anomalous diffraction (MAD) at the sulfur K-edge (5.02 A) with synchrotron radiation. The experiments were performed with uniaxial samples of oriented purple membranes at room temperature and 86% relative humidity. For such samples only the real part f' (lambda) of the resonant scattering amplitude of sulfur contributes to the observed scattering intensity. The sulfur density was determined from the difference in diffraction intensities detected at two wavelengths near the sulfur K-edge that were approximately 0.004 A apart. The measured change in f' between these two wavelengths corresponds to 6 electron units. This shows that large anomalous dispersion effects occur near the sulfur K-edge. The in-plane positions of the sulfur atoms of Met32, Met56, and Met209 were determined unambiguously. The difference density from Met20, Met60, Met118, and Met145 is concentrated in the interior of the seven alpha-helical bundle, overlaps strongly in the projected density map, and cannot be resolved at the resolution of these experiments (8.2 A). This method of localizing individual sulfur atoms can be applied to other two-dimensional protein crystals and is promising in conjunction with the site-directed introduction of sulfur atoms by the use of cysteine mutants.     

Experimental optimisation of the X-ray energy in microbeam radiation therapy

Microbeam radiation therapy has demonstrated superior normal tissue sparing properties compared to broadbeam radiation fields. The ratio of the microbeam peak dose to the valley dose (PVDR), which is dependent on the X-ray energy/spectrum and geometry, should be maximised for an optimal therapeutic ratio. Simulation studies in the literature report the optimal energy for MRT based on the PVDR. However, most of these studies have considered different microbeam geometries to that at the Imaging and Medical Beamline (50 μm beam width with a spacing of 400 μm). We present the first fully experimental investigation of the energy dependence of PVDR and microbeam penumbra. Using monochromatic X-ray energies in the range 40–120 keV the PVDR was shown to increase with increasing energy up to 100 keV before plateauing. PVDRs measured for pink beams were consistently higher than those for monochromatic energies similar or equivalent to the average energy of the spectrum. The highest PVDR was found for a pink beam average energy of 124 keV. Conversely, the microbeam penumbra decreased with increasing energy before plateauing for energies above 90 keV. The effect of bone on the PVDR was investigated at energies 60, 95 and 120 keV. At depths greater than 20 mm beyond the bone/water interface there was almost no effect on the PVDR. In conclusion, the optimal energy range for MRT at IMBL is 90–120 keV, however when considering the IMBL flux at different energies, a spectrum with 95 keV weighted average energy was found to be the best compromise.