Crystal Structure Theory and Applications, 2012, 1, 84-91
http://dx.doi.org/10.4236/csta.2012.13016 Published Online December 2012 (http://www.SciRP.org/journal/csta)
Enhancing Lab Source Anomalous Scattering Using Cr Kα
Radiation for Its Potential Application in Determining
Macromolecular Structures
Sibi Narayanan, Devadasan Velmurugan*
Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Maraimalai (Guindy) Campus,
Chennai, India
Received October 16, 2012; revised November 22, 2012; accepted November 29, 2012
ABSTRACT
Obtaining phase information for the solution of macromolecular structures is a bottleneck in X-ray crystallography.
Anomalous dispersion was recognized as a powerful tool for phasing macromolecular structures. It was used mainly to
supplement the isomorphous replacement or to locate the anomalous scatterer itself. The first step in solving macro-
molecular structures by SAD (single-wavelength anomalous diffraction) is the location of the anomalous scatterers. The
SAD method for experimental phasing has evolved substantially in the recent years. A phasing tool, 5-amino-2,4,6-
triiodoisophthalic acid (I3C—magic triangle), was incorporated into three proteins, lysozyme, glucose isomerase and
thermolysin using quick-soaking and co-crystallization method in order to understand the binding of metal ion with
proteins. Th e high qu ality of the diffraction da ta, the us e of chro mium anode X-ray radiation and the requ ired amount of
anomalous signal enabled way for successful structure determination and automated model building. An analysis and/or
comparison of the sulfur and iodine anomalous signals at the Cr Kα wavelength are discussed.
Keywords: Anomalous Scattering; SAD Phasing; I3C; Lysozyme; Glucose Isomerase; Thermolysin
1. Introduction
Current structural genomics projects aim to solve a large
number of selected protein structures as fast as possible.
High degree of automation and standardization is requir-
ed at every step of the whole process to speed up protein
structure determination. It is not easy to obtain auto-
matically the crystal d erivatives, which is appropriate for
phasing. New ideas have been put forward, that aim in
making the phasing of novel structures easier and more
susceptible to routine and automatic treatment [1]. Phase
problem is a bottleneck in macromolecular structure
determination and also in model building which is a
time-consuming task. Phases can be derived from some
knowledge of the molecular structure. Structures of small
proteins (molecular weight less than 10 kDa) can be
determined in solu tion using nuclear magnetic resonance
(NMR) spectroscopy and the assembly of proteins in a
complex can be studied using electron microscopy, but
only X-ray diffraction helps in determining the three
dimensional structure of small and large proteins with a
precision of about 0.1 - 0.2 Å. In macromolecular cry-
stallography, the phases are derived either by Molecular
Replacement (MR) method using the atomic coordinates
of a structurally similar protein or by locating the posi-
tions of heavy atoms that are intrinsic to the protein or
that have been added (MIR, MIRAS, SIR, SIRAS, MAD
and SAD) [2-5].
MIR is a classic method of solving novel crystal struc-
tures of macromolecules and has been responsible for an
enormous amount of success of structural biology, since
the early days of protein crystallography. MR also has
been widely used when appropriate models are available.
Over the past decade, MAD has been a vehicle of pro-
gress in phasing new crystal structures. Both MIR and
MAD require the presence of either appropriate heavy
atoms or anomalous scatterers, which are naturally oc-
curring or specifically introduced in the macromolecule
[6]. The standard method of derivatization in MIR in-
volves soaking or crystallizing the native crystals in di-
luted solutions or heavy metal reagents. In MAD, sele-
nium derivatization is carried out by genetic engineering
using which the normally occurring Methionines (Met)
are replaced as Se-Met. Both the approaches have draw-
backs because of heavy-atom derivatization, which re-
sults in non-isomorphism between the native and deri-
vatized protein crystals [7]. Sometimes, several deriva-
*Corresponding a uthor.
C
opyright © 2012 SciRes. CSTA

S. NARAYANAN, D. VELMURUGAN 85
tives are required to achieve success. By collecting Mul-
tiple wavelength anomalous diffraction (MAD) data at
two or more wavelengths, the definitive phase angle can
be determined using MAD technique [8,9].
In comparison, only a single set of X-ray data is re-
quired by Single wavelength anomalous diffraction tech-
nique (SAD) technique to provide the positions of the
anomalous scatterers, which together with density modi-
fication can reveal the structure of the complete protein.
The sulfur SAD phasing method allows the determina-
tion of protein structure de novo without reference to
derivatives such as Se-Met [10,11]. For targets, with a
weak MR solution from which structure cannot be de-
termined, the MR solution can be incorporated into the
SAD experiment and the increased number of sites iden-
tified by combined SAD/MR could be used in a subse-
quent SAD experiment with no MR component to calcu-
late the phases [12]. The number of protein structures be-
ing phased using only a single set of diffraction data by
SAD method has increased due to improved in-house
X-ray sources, detectors and softwares. This technique
has led to the routine use of anomalous scattering to ob-
tain phase information from either intrinsic sulfurs or
phosphorus presented in macromolecules or by addition
of heavy metal reagents by soaking /co-crystallizing method
in the native protein crystal [13,14]. In its purest form,
SAD can simply utilize the intrinsic anomalous scatterers
presented in the macromolecule, such as the sulfur atoms
of cysteine and methionine or bound ions [15]. The chal-
lenge is to maximize and measure the small signal, since
the Bijvoet ratio can be as low as 1% [16,17]. Both cop-
per and chromium anodes (wavelengths 1.54 Å and 2.29
Å) have been increasingly employed for the same pur-
pose in laboratory X-ray sources with much success
[18,19]. SAD phasing has already been carried out with
anomalous scatterers such as mercury [20], uranium [21],
iodine [22] and a tantalum bromide cluster [23], incor-
porated into the crystal lattice. However, heavy atom
derivatives suffer from nonspecific binding, which re-
sults in low occupancy of the heavy-atom sites, which
leads to weak anomalous signal and disruption of the
crystal lattice and fail in derivatization. Surprisingly, it
has been observed that short halide soaks can improve
the crystal diffraction [24]. Many such soak s also requ ire
the use of toxic chemicals and stringent safety precau-
tions [25]. Exploiting the anomalous signal already pre-
sented in the native protein or in the solvent would eli-
minate the extra experimental work via derivatization
and would also eliminate the risk of lack of isomorphism.
Phasing using the anomalous signal of sulfur alone has
earlier been achieved [26]. Longer wavelengths than Cu
Kα (1.54 Å) would produce a larger signal, but at the
same time experimental difficulties may increase as does
the noise level in the data [27]. It has been recently re-
ported that data collection wavelengths in the range of λ =
1.5 - 3.0 Å are fairly easy to handle in a diffraction ex-
periment and even at home sources using instant Cr Kα
radiation [28,29].
The use of chromium-anode X-ray radiation is very
useful for SAD experiments. The anomalous scattering
signal at this wavelength is more than doubled for vari-
ous metals when compared to conventional copper charac-
teristic wavelength. Furthermore, naturally bound metals
and atoms from crystallization solutions tend to show a
significant increase in anomalous scattering with chro-
mium radiation [30]. Improved data quality helps in ex-
ploiting the weak anomalous signal derived only from
the sulfurs or in particular from halide ions incorporated
by soaking. Therefore, a new class of compound 5-ami-
no-2,4,6-triiodoisophthalic acid, (I3C) that combines heavy
atoms for phasing with functional groups for their spe-
cific interaction(s) with biological macromolecules was
used to give rise to strong anomalous signals using in-
house Cr Kα radiation. The I3C consists of three iodine
atoms that are arranged in an equilateral triangle (6.1 Å
per side each) (Figure 1).
I3C has low toxicity when compared with other heavy
reagents. I3C has been incorporated into three proteins
viz., lysozyme (HEWL), glucose isomerase (GI) and ther-
molysin (TL) for the present study using quick-soaking
and co-crystallization method. Lysozyme and glucose
isomerase contain higher amount of sulfurs than most
proteins in the bacterial or eukaryotic proteomes provid-
ing a favorable Bijvoet ratio [31]. Thermolysin contains
lesser amount of sulfurs when compared to lysozyme and
glucose isomerase. I3C was derivatized successfully us-
ing soaking concentrations of 500 mM for lysozyme and
150 mM for glucose isomerase. I3C was also derivatized
into thermolysin using co-crystallization method. The
functional groups of the compound, interacts well with
the proteins via hydrogen bonds. The strong anomalous
signal of iodine atoms in the I3C makes it a powerful
phasing tool for in-house data.
2. Methods
2.1. Crystallization
The hen egg white lysozyme crystallization droplet
Figure 1. Graphical representation of I3C.

S. NARAYANAN, D. VELMURUGAN
86
consisted of 2 µl protein solution (20 mg/ml) and 1 µl
reservoir solution [50 mM Sodium Acetate and 1 M So-
dium Chloride, pH 4.7] and was equilib rated again st 1 ml
well solution at 25˚C. Glucose isomerase crystallization
droplet consisted of 2 µl protein solution (33 mg/ml) and
1 µl reservoir solution [200 mM Magnesium chloride and
100 mM Tris, pH 4.7] and was equilibrated against 1ml
well solution at 25˚C. Thermolysin crystallizatio n drop let
consisted of 2 µl protein solution (25 mg/ml), 1 µl reser-
voir solution [1.4 mM Calcium Acetate, 10 mM Zinc
Acetate, 1 mM Sodium Nitrate and 50 mM Tris; pH 7.3],
which was equilibrated against 1 ml well solution at 20˚C.
Lysozyme, Glucose Isomerase and Thermolysin crystals
appeared after a day and belonged to the tetragonal
P43212, orthorhombic I222 and hexagonal P6122 space
groups, respectively with one molecule per asymmetric
unit. Protein crystals were obtained using the hanging
drop vapour-diffusion method. Crystals of each protein
were harvested for collecting native and I3C quick-soak-
ed and co-crystallized datasets (500 mM I3C, 150 mM
I3C and 300 mM I3C).
Stock solutions of I3C with 1 M concentration were
obtained by dissolving so lid materials in 2 M lithium hy-
droxide solution [32] to deprotonate the carboxyl groups,
thereby producing a salt with high solubility. If sodium
or potassium hydroxide solution or an ammonia base is
used, the resulting salt will have limited solubility. Ly-
sozyme was soaked for about 45 seconds in 500 mM I3C
solution. Glucose isomerase crystal was soaked and tried
in 500 mM, 400 mM, 300 mM and 250 mM I3C for
various time-periods, but the crystal degr aded even tually.
Therefore, Glucose isomerase protein crystal was soaked
for about 3 minu tes 10 second s (190 seco nds) in 150 mM
I3C solution. The crystals were later back-soaked for 5
seconds in a cryosolution containing the same salt and
buffer concentration with 30% Glycerol and 25% MPD
(2-methyl-2,4-pentanediol) respectively. Thermolysin pro-
tein crystal was grown by adding 0.5 µl of 300 mM I3C
in the crystallization drop itself using co-crystallization
method. The grown protein crystals of thermolysin with
I3C were cryo-soaked in [10 mM Calcium Acetate, 7%
(v/v) DMSO, 20% (v/v) Glycerol and 10 mM Tris; pH
7.3]. The crystals were later flash cooled in liquid nitro-
gen (100 K).
2.2. Data Collection and Processing
Six datasets (native and 500 mM I3C for lysozyme; na-
tive and 150 mM I3C for glucose isomerase; native and
300 mM I3C for thermolysin) were collected separately
using Rigaku R-Axis IV++ image plate detector equip-
ed with Cr Kα (2.29 Å) anode X-ray generator operated
at 45 kV and 45 mA. Crystals diffracted upto 2.53 Å and
360 frames were collected with crystal to detector dis-
tance being 110 mm at 0.5˚ oscillation steps and 180
seconds exposure time per frame in each case. The inten-
sities were integrated with the HKL2000 [33], refining
all parameters including crystal mosaicity. Scaling and
mergin g we re a l s o done with the same package.
3. Results and Discussion
3.1. Substructure Solution and Data Analysis
The possibility of locating the anomalous scatterers using
the dual-space recycling algorithm enabled in SHELXD
depends on the signif icance of the anomalous signal pre-
sented in the data [34-36]. For the location of substruc-
tures of anomalous scatterers with SHELXD, only the
internal loop which relies on the strongest E magnitudes
is used. The success rate of SHELXD solutions critically
depends on data quality and redundancy of their mea-
surements. Using the direct methods program SHELXD,
it was possible to obtain th e positions of anomalous scat-
terers from the anomalous signal contained in all the dif-
fraction data. Density modification with SHELXE [37,38]
resulted in high-quality starting phases. Model building
was performed with ARP/wARP [39] and refinement
with REFMAC [40] available in CCP4i suite [41]. Fig-
ures were prepared using PyMOL software [42].
At the chromium wavelength of 2.29 Å, sulfur atom
has a value of 1.14, SHELXD program found seven sul-
fur atoms and eight chloride ions (weaker peaks ap-
peared in substructure solution), anomalous scatterers in
the native lysozyme data (Figure 2(a)). Chlorine, the
halide lighter than iodine, has its K edge at a long wave-
length (4.39 Å) and disp lays only a small anomalous effect
[43-46]. For native glucose isomerase data, anomalous
scatterers for one manganese and nine sulfur atoms were
obtained using SHELXD program and treated for phas-
ing (Figure 2(b)). Similarly, for native thermolysin data,
anomalous scatterers for one zinc ion, four calcium ions
and two sulfur atoms were obtained using SHELXD pro-
gram and treated directly for phasing (Figure 2(c)). They
were given as input into SHELXE for obtaining the elec-
tron density maps. The density modified final maps were
subjected to analysis by ARP/wARP web server [47] for
automatic chain tracing and model building. The electron
density map allowed ARP/wARP program to build 122
residues out of a total of 129 amino acids for native ly-
sozyme with four disulfide bridges. Similarly, 388 resi-
dues were automatically built o ut of a total of 389 amino
acids for native glucose isomerase with a single disulfide
bridge. For native thermolysin, ARP/wARP program au-
tomatically built 314 residues out of a total of 316 resi-
dues.
The iodine absorption edges retain a significant
anomalous signal (f" = 12.82 e) at the chromium charac-
teristic wavelength (2.29 Å). For I3C-soaked lysozyme
dataset, substructure solution determined heavy-ato m site

S. NARAYANAN, D. VELMURUGAN 87
for iodines of I3C that formed an equilateral triangle and
anomalous scatterers for eight sulfur atoms and eight
chloride ions (Figure 3(a)). For I3C-soaked glucose
isomerase dataset, anomalous scatterers for twelve sulfur
atoms, one manganese ion and for an equilateral triangle
(a)
(b)
(c)
Figure 2. Anomalous map at 5 sigma level. (a) the peaks of
seven sulfur atoms (big) and eight chloride ions (small) with
water molecules in HEWL; (b) the peaks of nine sulfur at oms
and one manganese ion with water molecules in GI and (c)
peaks of four calcium ions, one zinc ion and tw o sulfur atom s
with water molecules in TL.
(a)
(b)
(c)
Figure 3. Anomalous map at 5 sigma level. (a) the peaks of
one I3C molecule and eight sulfur atoms (big) and eight
chloride ions (small) with water molecules in HEWL; (b)
the peaks of one I3C molecule and twelve sulfur atoms and
one manganese ion with water molecules in GI; and (c)
peaks of two calcium ions, one zinc ion, two sulfur atoms
and one I3C molecule with water molecules in TL.
(I3C) were determined using SHELXD (Figure 3(b)).
Using SHELXD program, for I3C co-crystallized ther-
molysin dataset, anomalous scatterers for two calcium
ions, one zinc ion, two sulfur atoms and one I3C mole-
cule were determined (Figure 3(c)). Density modifica-
tion was carried out using SHELXE and the obtained
modified map was given as input into ARP/wARP pro-
gram. The final ARP/wARP conventional and free R

S. NARAYANAN, D. VELMURUGAN
88
factors obtained with REFMAC were 20.3% and 22.4%
(lysozyme); 21.4% and 24.9% (glucose isomerase); 20.1%
and 22.4% (thermolysin) respectively, wherein 123 resi-
dues of 129 amino acids for lysozyme, 386 residues of
the total of 389 protein amino acids for glucose isom-
erase and 312 residues out of 316 amino acids for ther-
molysin were safely built by the iterative free-atom den-
sity modification and model-building procedure. Only
halide sites corresponding to peaks higher than 5σ in the
anomalous map were included.
Solvent content of lysozyme, glucose isomerase and
thermolysin are 37%, 55% and 46%, respectively. In all
the cases discussed above, it was possible not only to
locate the anomalous scatterers, but also subsequently to
solve the protein model by SAD phasing. All the col-
lected datasets are of good quality and th ey have close to
100% completeness. In all the above-mentioned struc-
tures, the asymmetric unit contains only a monomer.
Longer wavelengths provide not only an increased ano-
malous signal for phase determination, but also allow a
much clearer definition of substructures; their positions
and occupancies, which may turn out to be very impor-
tant for elucidating the function of a molecule.
3.2. Native Sulfur Binding Sites
By contrast, sulfur is presented in almost all proteins. It
is heavier than any other elements (C, N, and O) found in
most proteins and displays some anomalous signal. Phas-
ing a protein through only the inherent anomalous signal
derived from the sulfur atoms presented in both cysteines
and methionines presented in the ordered solvent region
was possible for lysozyme and glucose isomerase data
sets with redundancy of 10 and above at wavelength of
2.29 Å. The structure of the 129 residue, tetragonal ly-
sozyme (P43212) was phased using only the anomalous
signal derived from seven sulfurs in the protein and eight
coordinated chloride anions with high redundancy. Simi-
larly, the structure of orthorhombic glucose isomerase
was phased using one manganese and nine sulfur atoms
from eight methionines and one cysteine from the 389
residues presented in the protein. The structure of 314
residue, hexagonal thermolysin (P6122) was phased using
the anomalous signal derived from two sulfur atoms, one
zinc ion and four calcium ions.
The presence of metal ions originated from the crys-
tallization buffer used for each protein crystallization
setup.
3.3. I3C Binding Sites
One binding site for I3C each was observed in lysozyme,
glucose isomerase and thermolysin, respectively. The oc-
cupancies for all three halogen atoms per site were (0.70,
0.66 & 0.62) for lysozyme (0.65, 0.60 & 0.56) for glu
cose isomerase and (0.81, 0.77 & 0.71) for thermolysin.
Interestingly, the occupancy values of both the proteins
differ lightly, although similar soaking conditions were
tried. The data from the I3C derivative showed signifi-
cant anomalous signal to noise ratio (1.78% for lysozyme,
1.82% for glucose isomerase and 1.45% for thermolysin)
throughout the entire resolution range to 2.53 Å. The
interactions of the I3C in lysozyme are very similar to
those previously reported [21]. They mostly replace wa-
ter molecules in the crystal lattice. Inspection of the I3C
sites using PyMOL software showed several Hydrogen
bond interactions. The three functional groups of the
phasing molecule formed hydrogen bonds with the side
or the main chains of the amino acids. One carboxyl
group interacts with an Arginine residue (ARG 114). The
same carboxyl group interacts with oxygen and nitrogen
atoms of the Asparagine residue (ASN 37) (bifurcation)
via water molecules. Hydroxyl group of the I3C mole-
cule also interacts with the nitrogen presented in the Ly-
sine residue (L YS 33) (Figure 4(a)).
(a)
(b)
(c)
Figure 4. I3C binding site and interactions (a) HEWL; (b)
GI; and (c) TL.

S. NARAYANAN, D. VELMURUGAN
89
aked glucose isomerase data, the amino
gr
w
se
Table 1. Crystal data statistics, phasing and model building details.
Native
L Thin ed Gl I3C-Cocrystallized
In the I3C-so
oup of I3C forms hydrogen bonds to the hydroxyl
group presented in the Phenylalanine residue (PHE 296).
Similarly, the amino group also shows an interaction
ith Glycine residue (GLY 298) via a water molecule.
The carboxyl group interacts with the oxygen atom of the
Aspartic acid residue (ASP 295) (Figure 4(b)). In the
I3C co-crystallized thermolysin data, the hydroxyl group
of I3C forms a hydrogen bond with Serine residue (SER
279) (Figure 4(c)). Sulfur atoms could also be located in
the 500 mM I3C, 150 mM I3C and 300 mM I3C datasets
collected to 2.53 Å resolution using Cr Kα radiation.
Crystal data statistics, phasing and model building details
are listed in Table 1. In all cases, the figure of merit was
greater than 0.55. More than 96% of the residues and
92% of the side chains were placed automatically using
warpntrace mode in ARP/wARP program. Anomalous
difference Fourier maps have been computed at 5σ level.
The concentration of iodides in the soaking solution
ems to influence their occupancy more significantly.
Iodide sites in the I3C ring have hydrogen bonding con-
tacts with hydrogen-donor groups of protein or water
molecules. They tend to occupy ordered sites around the
protein surface with varying occupancy, and therefore
share with water molecules presented nearby. This shows
that I3C has easily diffused into the protein crystals dur-
ing quick-soaking and co-crystallization methods. The
quick cryo-soaking and co-crystallization methods with
halides explained here may be an alternative method for
phasing protein crystal structures.
4. Conclusion
Data quality is decisive for successful location of the
anomalous substructure. The example of successful SAD
phasing based on the signal of weak anomalous scatterers
such as sulfur atom and chloride ion, prove that even the
anomalous signal provided or presented naturally in a
macromolecule is good enoug h to solve crystal structures
successfully using an in-house chromium-generated X-
ray radiation. The results also indicate that phasing after
a short soak with a buffer containing a halide salt or
co-crystallization is much easier and more likely to suc-
ceed. I3C represents a novel class of compound that
helps in showing interaction(s) with protein molecule(s),
it can be used for experimental phasing, and is a com-
pound of choice, since the iodine atoms give rise to a
strong an omalous signal fo r S AD phasin g.
ysozyme Native Glucose Native I3C-Soaked I3C-Soak
Isomerase ermolys Lysozyme u cose Is omeraseThermolysin
Cell Parameters a =,
α ˚ α
aA ,
α ˚α
a
α = 20˚
b = 77.67 Å
c = 37.43 Å
= β = γ = 90.0
a = 92.78 Å,
b = 97.31 Å,
c = 102.64 Å
= β = γ = 90.0˚
= b = 92.76 Å,
c = 128.36 Å
α= β = 90.0˚,
γ = 120˚
= b= 77.96 Å
c = 37.25 Å
= β = γ = 90.0
a = 93.01 Å,
b = 97.20 Å,
c = 102.41 Å
= β = γ = 90.0˚
= b = 92.73 Å,
c = 128.48 Å
β = 90.0˚, γ = 1
Space Group 3 13 11
Resolution Range 27.48 Å -53 Å 27.48 Å -53 Å38.33 Å -53 Å27.48 Å -53 Å27.48 Å -53 Å 40.15 Å -.53 Å
S
4117 314139 31
10. 10. 43.49 ( 10. 12 . 40.
Co)
Anomignal
Map Cor
Total Residues Built
wor k
Nument
Molecules
P4 22 I222 P6122 P422 I222 P622
Mosaicity 0.76 0.49 0.6 0.56 0.53 0.49
2. 2. 2. 2. 2.2
olvent Content (%) 37 55 46 37 55 45
Unique Reflections 6759 1449 5870 1433
Redundancy 6 (9.98)3 (9.73)42.84)3 (9.73)8 (10.11)61 (40.12)
mpleteness (%99.5 (97.8) 98.1 (94.8) 100 .0 (100.0) 98.7 (95.6) 97.7 (93.6) 99.9 (100.0)
I/σ(I) 57.6 (13.3) 17.4 (4.8) 19.9 (7.1) 56.9 (14.8) 16.8 (4.2) 23.0 (7.3)
alous S
(%)
FOM
0.0171 0.0183 0.0146 0.0178 0.0182 0.0145
0.56 0.58 0.53 0.57 0.61 0.58
relation 0.76 0.81 0.79 0.78 0.79 0.80
BWilson 10.53 12.79 39.69 11.08 13.52 33.24
122 388 314 123 386 312
Side Chains 110 383 311 114 383 310
R (%) 24.4 21.3 18.6 20.3 21.4 20.1
Rfree (%) 28.6 24.8 21.9 22.4 24.9 22.4
ber of Solv102 140 145 154 128 156

S. NARAYANAN, D. VELMURUGAN
90
5 emen
overnment of India for the
arch. DV thanks DST-FI ST
ENCES
[1] W. A. HendriPhase Determina-
tion from Mus Diffraction Mea-
. Acknowledgts
SN and DV thank UGC, G
financial suppor t for this rese
and UGC-SAP for funding facilities to the Centre for
Advanced Study in Crystallography and Biophysics.
Chromium datasets were collected at X-ray facility,
CCMB, Hyderabad funded by CSIR Facility Creation
Project (FAC0004) as part of Eleventh Five Year Plan.
SN and DV thank Dr. R. Shankaranarayanan for extend-
ing his lab facilities to collect anomalous scattering d ata-
sets using Cr Kα radiation.
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