X-ray energy selection <i>via</i> direct fluorescence measurements.



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X-ray energy selection via direct fluorescence measurements.

The iridium derivative crystal used in the screening had already been subjected to several hours of exposure to X-rays. For this reason a fresh crystal taken from the same batch was used for the MAD work. The crystal had dimensions , the shortest side pointing along the c axis. The crystal was a bipyramidal prism except for a small corner which had been chipped off during mounting.

The choice of X-ray energies for the experiment was made on the basis of fluorescence measurements made across the Iridium absorption edge recorded from the derivative crystal. This was done on X31 using the fluorescence apparatus in conjunction with the energy calibrator described in Chapter gif to obtain experimental data of the the edge fluorescence on an absolute energy scale. A Si(311) monochromator was used to obtain an energy resolution of at the Iridium edge.

The results of the calibration for this data are represented in Fig. gif which shows the calibration curve obtained by plotting the discrepancy between the calculated and approximated energies of the reflection against the latter value. The estimated error in the calibration of the energy axis is . After correcting the experimental fluorescence data to place it on an absolute scale, the procedure described in Sec. gif was used to produce the curves of and shown in Fig. gif.

  
Figure: Calibration curve produced by the absolute calibration procedure for the fluorescence data over the Iridium edge in lysozyme. The differing error bar sizes indicate the degree of reliability in the reflections depending on whether they are scattered more in the horizontal plane where the larger horizontal beam divergence tends to broaden the reflections making it more difficult to accurately determined the centroids of the reflections or in the vertical plane where the reflection have smaller widths.

  
Figure: Plot of and against energy around the iridium absorption edge calculated from X-ray fluorescence measurements on an iridium derivative of crystalline lysozyme. The noise in the experimental fluorescence data was on the level of about . The data were treated as described in the previous chapter to produce spectra of (upper curve) and . The estimated error in the calculation of the values is e which may be introduced as a result of the spline fitting routine in the above edge region. Data sets 2, 3, 4 and 5 were measured at the X-ray energies labelled in the diagram.

The curve (upper) shows a large white line at the absorption edge which has a maximum of . The rising and falling inflection points on the white line have the effect of creating a local minimum and maximum in the curve. The value of in this near edge region changes from to over with the maximum lying within this range.

  
Table: Diffraction data from the iridium derivative of lysozyme was collected at five X-ray energies at and around the Iridium absorption edge using the X31 beam-line (1-5) and at two more energies using the X11 beam-line (6-7). The table lists these energies along with the values of the experimentally determined anomalous scattering factors for iridium. ( indicate values calculated using the program CROSSEC). No direct measurement of and was possible for data set 7 and values from CROSSEC are not applicable for the region around an absorption edge.

The energies chosen for MAD experiment are shown in Table gif. Measurements 1 to 5 were performed using X31. Data sets 1 and 2 were measured at energies above and below the edge so as to obtain data with a low contribution (2) and a low contribution (1). At both of these energies the anomalous scattering factors are slowly varying and are not sensitive to instabilities in the X-ray energy. The values of the anomalous scattering factors were therefore well defined and were expected to provide a useful control with which to assess the performance of the calibrator at the other more sensitive X-ray energies by comparing the observed anomalous signals for all data sets. Data sets 3, 4 and 5 were measured within the region at the edge so as to take advantage of the prominent white line [94] [62]. Data sets 6 and 7 were measured on the X11 line at energies which did not take advantage of the sharp features in the absorption spectra but were aimed at obtaining as much contrast in the anomalous scattering factors as possible given the large bandpass.

The effect of measuring at these X-ray energies on the atomic scattering factor of the iridium may be visualised with the help of Fig. gif which shows the path traced by the end of the complex scattering vector as the energy is changed across the absorption edge. Following the line clockwise in the upper half of the diagram is equivalent to increasing the energy across the edge. The upper plot is reflected in the real () axis so as to represent the contribution of the iridium scattering factor to a Friedel mate. Phillips [94] showed that in theory increased phasing power from a set of measurements is achieved when the points in Fig. gif are positioned as far away from each other as possible, the ideal case being when for example three points form the vertices of an equilateral triangle.

  
Figure: Plot of against over the iridium edge in crystalline lysozyme. The five points present in the figure represent the energies chosen for the experiments on X31. The energies chosen maximise the contrast in the iridium anomalous scattering factors for the limited energy range represented.



next up previous contents index
Next: Data collection. Up: MAD experiments on Previous: Derivative screening.



Gwyndaf Evans
Fri Oct 7 15:42:16 MET 1994