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 
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. 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. was used to produce the curves
of 
and 
shown in Fig. .
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 .
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. 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.
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.