Research and Reviews: Journal of Dairy Science and Technology

Volume 2, Issue 1, ISSN: 2319-3409

__________________________________________________________________________________________

X-Ray Crystallography and Its Applications in Dairy

Science: A Review
Kamal Gandhi*, Anil Kumar, Saurabh Gosewade, Ravinder Kaushik, Darshan Lal
Dairy Chemistry Division, National Dairy Research Institute, Karnal, Haryana, India, 132001

Abstract
X-ray crystallography (XRC) is the study of crystals using X-rays. XRC is the primary
method in which detailed structure of molecules especially molecules that pertain to
living systems have been visualized and discovered by exposing a well-ordered crystal of
a substance to X-rays and finally generating the structural information from the spots
produced on a film due to this impact. X-ray crystallography has been used for analysis
of liquid milk, milk powders, milkstones, polymorphism of milk fat and most widely and
importantly in discovering the structure of most of the milk proteins and thus helping in
correlating their structure with possible functions.

Keywords: X-ray crystallography, crystals, detector, resolution, electron density

maps

*Author for Correspondence E-mail: [email protected], Tel: + 91-7206270904

INTRODUCTION experiments. Production of suitable crystals is

X-rays have short enough wavelengths to rate limiting for study of many proteins [1].
“see” the atoms and the molecular structure of High purity of protein preparations used for
molecules. Any structure can be visualized crystallization is the most important factor for
only if electromagnetic radiation of a growing diffraction quality crystals.
wavelength comparable to its dimensions is
used. For proteins, appropriate size is of the X-RAY CRYSTALLOGRAPHY/
order of Å (10−10 m). Wavelength of X-rays (1 X-RAY DIFFRACTION
to 100 Å) approximates to this; hence, use of It is a technique which relies on the dual
X-ray crystallography technique is useful for nature (wave/particle) of X-rays to discover
studying structure of biological information about structure of crystalline
macromolecules. materials. In this technique, the pattern
produced by the diffraction of the X-rays
X-ray crystallography is the most widely used through the closely spaced lattice of atoms in a
technique to obtain high-resolution protein crystal is recorded and then analyzed to reveal
structural information. It is the study of the nature of that lattice. This technique entails
crystals using X-rays. XRC is the primary bombarding a sample of protein in crystalline
method in which detailed structure of form with a beam of X-rays. Most of these X-
molecules especially molecules that pertain to rays pass straight through the crystal but some
living systems have been visualized and are diffracted by it, resulting in a diffraction
discovered by exposing a well-ordered crystal pattern recorded on a detector (Figure 1). This
of a substance to X-rays and finally generating diffraction pattern is a reflection of three-
the structural information from the spots dimensional structure of protein molecule
produced on a film due to this impact. present in the crystal. X-ray crystallography
can provide very detailed atomic information,
Crystals are not a requirement of analysis showing every atom in a protein or nucleic
using X-rays; any ordered (or partially acid along with atomic details of ligands,
ordered) array of molecules can produce useful inhibitors, ions, and other molecules that are
X-ray data. But, crystals are the most incorporated into the crystal.
favorable samples. So, crystals have become
the prerequisite for X-ray diffraction

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 X-Ray Crystallography and Its Applications in Dairy Science Gandhi et al.
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Fig 1: Schematic of X-Ray Diffractometer.

Basic Method of X-ray Crystallography X-ray source is usually a sealed tube in which
electrons are accelerated from one end and
allowed to impinge at other end on a metal
target, usually copper or molybdenum for
biologically relevant samples. This produces
X-rays of wavelength 1.5418 Å (for Cu) and
0.7107 Å (for Mo) (Figure 3).

Fig 2: X-Ray Crystallography Method to

Obtain 3D Structure of Molecules.

Crystallize the molecule, subject it to

irradiation by a beam of X-rays and make a
record of the 3D diffraction pattern. Analysis
of this data through computer calculations Fig. 3: X-Ray Generation Using X-Ray
results in a model of molecule which can then Vacuum Tubes.
be refined against observed X-ray data.
Finally, one obtains 3D coordinates which Diffraction by X-rays is described in terms of
describe position in space of each atom in the reflections from crystal plane. Diffraction
molecule (Figure 2). Such a refined model can occurs when two or more waves are combined.
be displayed and manipulated either as plastic
or wooden models or on a computer graphics Bragg’s Law
format. Bragg’s law is a fundamental law and is valid
for monochromatic X-rays only and is used to
Generation of X-Rays calculate interplanar spacing used in XRD
X-rays can be generated when a metal plate is spectra. It defines the spacing (d) of atomic
bombarded with accelerating electrons. This is planes and incident angle (θ) at which X-rays
normally achieved in high voltage tubes, but of a particular wavelength will reflect in phase
more powerful X-ray beams may be generated (i.e., diffract) (Figure 4). For constructive
in synchrotron storage rings, using electrons interference to occur, difference in path
traveling with the speed of light. lengths given by BC + BD should be equal to
an integral number of wavelength of the
incident beam.

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 Research and Reviews: Journal of Dairy Science and Technology
Volume 2, Issue 1, ISSN: 2319-3409
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Fig. 4: Bragg’s Reflection Condition or Bragg’s Law.

BC = BD = d sinθ 6. Further refine the position of heavy atom

2d sinθ = n using least square of difference Fourier
technique.

 = wavelength of the X-rays

where, n = an integer 7. Using estimated phases and observed
amplitudes of each F (h, k, l), one can

 = diffraction angle
d = interplanar spacing in the specimen create an electron density map.

sinθ  1/d (reciprocal space)

8. A model is built of electron density map.
As low resolution data (about 5.5–7 Å) is
used, so map does not show well resolved
When a crystal is irradiated by an X-ray beam, structural details.
Bragg’s law is used to predict the angle θ at
which diffracted intensities may be expected.  Then, step 4 and 8 are repeated at higher
This may require the knowledge of inter- resolution (2.5–3 Å) until it is possible to
planar spacing d and of X-rays. Conversely, construct a molecular model.
if angles of occurrence (and relative 9. Further refine the structure using Fourier
intensities) of diffracted spots are measured in or least square technique and can also
an experiment, corresponding inter-planar include information about known
spacing in crystal may be calculated and lattice energetic of protein conformation.

‡ Experimentally determined 3D structure

structure can be determined. 10. Check R-factor of the models.

Steps to Determine Structure of Molecules information is stored in various databases

‡ Principal of such database is Brookhaven

1. Prepare suitable crystals of native as sets of atomic coordinates.
macromolecules. Using crystals, one can
determine the space group and then collect protein database (PDB) which may be
a set of scattering amplitude data. accessed via internet at
2. Prepare several different heavy atom http://www.pdb.bnl.gov. There are more
isomorphous derivatives (having same unit than 12,000 sets of crystal structure
cell, space group and macromolecular coordinates for a wide variety of proteins

‡ Various internet sites provide free

structure as parent crystal, except that one in this data bank.
or more heavy atoms have been introduced
at specific loci). For each derivative, software programs which can generate
collect a new set of scattering amplitude protein structural models from atomic
data. coordinates. These structures can be
3. Attempt to find location of heavy atoms in viewed interactively (e.g., rotated and

‡ RasMol
crystals. zoomed).
4. Refine the positions using Fourier
refinement technique. (http://www.umass.edu/microbio/rasmol)
5. Phases of each F (h, k, l) can be estimated is amongst such best known programs.
comparing the structure factor data of Another program termed Mage provides
parent crystal seven heavy-atom interactive protein structural models called
isomorphous derivatives. “Kinemages”

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 X-Ray Crystallography and Its Applications in Dairy Science Gandhi et al.
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‡
‡
(http://www.fascb.org/protein/kinemage/ki Convection

‡
npage.html). Volume of crystallization

‡
Pressure, vibrations
Crystallization Additives, their character and

‡ Solvents (especially for small molecules)

Although many low-molecular-weight concentration
substances crystallize relatively easily, this is
not the case for extremely larger ones which To grow crystals of any compound from
display irregular surfaces. Even if induced to solution, molecules have to be brought to a
crystallize, protein crystals will contain supersaturated state. When solution returns to
solvent-filled channels or pores between equilibrium, substances will precipitate out,
individual protein molecules. Only a small hopefully as crystals, not as an amorphous
proportion of surface of individual proteins precipitate. By varying one of the parameters
interacts with each-other and as a result, listed above, one may change solubility and
crystals are soft and easily destroyed. hence saturation level of molecules. In order to
Production of suitable crystal is rate limiting obtain good defect-free crystals of a size
for study of many proteins. suitable for X-ray analysis, equilibration needs
to be carefully controlled. A crystal is formed
Growth of Crystals of Biological Molecules when protein molecules are precipitated very
The chief difference between crystals of slowly from supersaturated solution and this is
biological substances and those of other usually achieved by slow evaporation, vapor
important materials such as silicon is that in diffusion or dialysis. Final condition of
the former, repeating units are invariably crystallization should be such that the
molecules or groups of molecules. crystallized state is thermodynamically most
Components required for While in latter, stable. Crystal growth is not well understood
repeating units are atoms or groups of atoms. and almost entirely empirical rules are
So, forces of bonding between one repeating followed to grow crystals.
unit and another are much weaker in former
being Vander Waal’s forces or hydrogen Methods of Crystallization
bonds. In latter, crystals are built up from Slow Evaporation Technique
strong covalent or ionic bonds. Crystals of Slow evaporation technique is conceptually
biological materials are thus usually soft and the simplest technique to grow crystals from
brittle than inorganic substances. Another supersaturated solutions. It is chiefly used to
difference is size – biological and organic crystallize small organic molecules. Solution is
molecules usually form very small crystals placed in a small glass beaker and sealed with
(approximately 1 mm3) as compared to an airtight cover, except one or two small
crystals of inorganic materials (approximately apertures through which slow evaporation of
1 cm3). solvent takes place bringing the solute to
supersaturation and hence to crystallization
Biological molecule crystals are finicky: some (Figure 7).
form perfect, well-ordered crystals and others
form only poor crystals. Biocrystals are
usually grown from solution and may have a
large solvent content [2]. This is especially
true for crystals of macromolecules such as
proteins and nucleic acid, where solvent can
take up as much as 80% crystals. Growth of
biocrystals is a multi-parameter process.

Factors Affecting Crystallization of Organic Fig. 5: Slow Evaporation Method of

Crystallization.
‡ Purity of sample
Molecules and Biomolecules

‡ Concentration Vapor Diffusion

‡ Temperature, pH of solution It is the most widely and successful method
‡ Time, rate of equilibration used for producing diffraction quality crystals.
‡ Ionic strength Vapor diffusion method (Figure 6)

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 Research and Reviews: Journal of Dairy Science and Technology
Volume 2, Issue 1, ISSN: 2319-3409
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a. Hanging drop method Though this may lead to a large number of tiny
b. Sitting drop method crystals, it frequently gives rise to good
A droplet of solution containing the molecule crystals.
is equilibrated against a reservoir containing
the precipitant (i.e., the crystallizing agent at a A protein drop can either be suspended from
higher concentration than in the drop) and is the cover slip used to seal the reservoir (a
slowly dehydrated in a sealed well by hanging drop) or set on some sort of support
equilibration with a reservoir at higher above the reservoir (a setting drop). Usually,
precipitant concentration. In batch methods, the drop is made by mixing equal volume of
the molecule to be crystallized is mixed with protein solution and reservoir solution.
crystallizing agent at a high concentration so
that supersaturation is immediately reached.

Hanging Drop Method Sitting Drop Method

Fig. 6: Vapor Diffusion Methods.

Vapor diffusion method has the benefit of solution is generally layered over the solution
automatically screening a range of of molecule. Crystals usually grow at
precipitation conditions and has a defined end- interphase. Sometimes, seeds are introduced to
point. This should allow one to optimize induce the crystals to grow. These seeds may
crystallization condition so that the precipitant be extremely tiny crystallites.
concentration slowly increases just fast enough
to compensate for the loss of protein prior to Dialysis
crystallization. This requires optimization of This method has also been quite successful.
end-point of experiment and rate of Precipitant conditions are automatically
equilibration. screened, but unlike previous methods, protein
To vary the rate of equilibration is to vary concentration remains constant. Semi-
surface to volume ratio (i.e., size) of the drop. permeable membranes are used to contain
In general, larger drops will equilibrate more protein solution in thick-walled capillary tubes
slowly than smaller drops. However, a general or micro-dialysis buttons. Device is suspended
observation is that long equilibration times or immersed in a stepwise manner, with an
give better quality crystals. equilibration interval of a few days between
steps. This method may lead to crystal growth
Interface Diffusion at a particular height and aid choice of ideal
It is used for small molecules as well as for precipitant strength. But, dialysis methods are
large biological molecules. Precipitation not economical for proteins (Figure 7).

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Fig. 7: Dialysis Experiment.

Table 2: Components Required for Crystallization of Biomolecules.

Molecule Solvents Additives Precipitants
Small organic Organic solvents (e.g., Metal ions, salts Alcohol, organic
molecules CCl4, ethyl acetate, etc.), solvents
alcohol, water

Protein Aqueous solution Cofactors, sugars, Ammonium sulphate,

metal ions, polyethylene glycol
complexes, (PEG), methyl pentane
polyamines, salts diol (MPD)
Nucleic acids Aqueous solution Polyamines, metal MPD, PEG, isopropanol
ions, salts, etc. and other alcohols.

Micro-heterogeneity in Crystals now almost exclusively used in collection of

Micro-heterogeneity may occur due to data from crystals of biological
presence of some impurities or some local macromolecules.

 Outcome of data collection process is a list

aberrations (dislocations, changes in crystal
lattice, and modifications in protein structure)
which are visible in the crystal leading to of angles, indicating orientations of the
damage in crystal structure and result in crystal and the detector and intensity of
relatively weak diffraction patterns. Micro- reflection from oriented lattice plane at

 Instead of angles, it is usual to identify the

heterogeneity can have deleterious effects for this position.
crystal lattice and its solubility properties and
protein might become blurred by such set of planes that give rise to each
variability, leading to difficulties in defining diffracted spot by Miller indices hkl and
crystallization conditions. associate each value of hkl to the
appropriate value of intensity. From the
Detector test of intensities, amplitude of each
Detector of the diffracted X-rays may be a reflected wave is extracted by the simple
film, or a radiation counter. Films allow process of taking the square root of, since
recording of several reflections at a time, but the measured intensity of a wave is square
suffer from requirement of long exposure of its amplitude. These amplitudes,
times, as well as imprecision in conversion of represented as Fhkl, are now used in
blackening of film into a numerical value of analysis of the structure of molecules that
intensity. make up the crystal.
Modern devices such as multi-wire
proportional counters or imaging plates offer
advantages of both films and counter and are

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 Research and Reviews: Journal of Dairy Science and Technology
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‡
‡
Structure of Molecule Least square method

‡
Structure of a molecule is obtained by a Constrained-restrained refinement
Fourier transform of the observed amplitudes Using computer graphics
Fhkl. Fourier transform involves decomposition
of a complex periodic function (thick lines) Resolution of an X-Ray Structure
into its sine and cosine components (thin • Resolution is another model to see
lines). As more component waves at different how good your model is. Resolution
frequencies (wavelengths) are added, resulting gives the size of the smallest molecule
wave approaches a square wave more and you can see or resolve. It is dependent
more closely. Fourier decomposition of a on the amount of data ultimately
square wave would result in components of all phased and used in structure
possible frequencies. determination. Resolution can be
expressed as angular limit at which
It is not only necessary to know amplitudes internal data can be observed by
(and wavelengths) of simple waves, but also to Bragg’s law:
know the relative positions, or phases of waves 2d sinθ = n
in order to reconstruct original periodic i.e., lower the interplanar spacing d, greater the
function. resolution and higher the angle at which
diffracted beam corresponds to that set of
To reconstruct the crystal as a 3D planes is observed. Highest resolution
superposition of these simple waves of achievable using radiation of a particular
electron density, it is necessary to know the wavelength is d = /2 Å.
phases. Once phases are known, the
mathematical operation of Fourier transform,
 The non-contact, non-destructive
Advantages of XRD Technique
which corresponds to physical superposition of
simple waves, will yield a picture of nature of X-rays is ideally suited as a
distribution of electrons within the unit cell of
 Using diffracted X-rays, knowing the
probe to characterize materials.
the crystal. In other words, we get a picture of
structure of molecules in a unit cell. wavelength and diffraction angle, one
In an XRD experiment, phases cannot be can make use of Bragg’s law, to
measured directly and they have to be extract information on the crystalline

‡ Heavy atom or multiple isomorphous

determined using indirect methods such as: condition of the sample, and thus
phase proportions; texture and degree
replacement (MIR) method of preferred orientation can be
Some direct methods are also available calculated; crystal structure can be

‡ Molecular replacement method

such as: refined using appropriate

‡ Anomalous scattering technique  Plastic strain and particle size can also
mathematical models.

 A more recent solution to the phase

be calculated using peak width.

problem involves using synchrotron Limitations of XRD Technique besides

 Crystallizing Protein:
radiation at multiple wavelengths. This Phase Problem

‡ Fragile
has greatly accelerated the rate of solving

‡ Requires a crystal with shortest side

crystal structures.

‡ Crystallization may require conditions that

Refinement of Structure 0.2 mm
Once approximate values of phases are
determined, next step is to refine them. This is
 Flaws of Crystallization:
cannot be said to be physiological.

‡ Disorder in Unit Cell

more conveniently done by performing Fourier

‡ Vibrations of molecules
transform and refining the approximate

‡ Distortion in crystallization
positions of atoms obtained by including other
known data such as stereochemistry. For
refinement of the structure obtained by above
processes, following methods can be used:

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 Diffraction peaks are very crowded for properties of molecules, and may also provide
complex macromolecules, and are difficult clues about their possible role(s), that is, their

 Heavy atom substitution works for

to separate. function, in the organism. They can be thus
especially valuable tools for investigating
molecules larger than 600 atoms, so a gap structure-function relationships. X-ray
is present for molecules in the crystallography is an indispensable tool for
intermediate size range. molecular modeling. Select a target molecule,
purify it, and determine structure using
The principal types of information that can be suitable technique (e.g., XRD) and computer
secured by proper interpretation of X-ray data software program, compare the results with

‡ Crystalline or non-crystalline substances

are: suitable library of structures and get the

‡ Crystallographic
results. Increasingly, modeling software is
system, unit cell available for a variety of industrial

‡ Deduction of crystal unit (atom, ions or

dimensions applications.

‡ Chemical
molecule) Applications of X-Ray Crystallography in
identity, chemical and Dairy Science

‡ Allotropic changes
crystallographic changes X-ray crystallography technique has been a

‡ Single crystal or aggregate

widely used tool for elucidation of compounds

‡ Type and mechanism of alloy formation

present in milk and other types of information

‡ Random or fibered aggregate and relative

obtained through structure function
relationship. Although more detailed

‡ Grain size in an aggregate (in colloidal

degree of preferred orientation information from X-ray analysis has been
secured from substances which are commonly
known to be crystalline, it has been surprising
‡ Internal strain or distortion
range)
to find substances commonly thought of as
Basic applications of XRD technique being non-crystalline as actually having a
partially crystalline structure and that this
‡ Find structure to determine function of
involve:
structure can be changed by heat treatment,
pressure, stretching, etc. Casein is an example
‡ Distinguish between different crystal
protein
of the latter class of proteins. Stewart [3] has
shown that even solutions tend to assume an
‡ Study crystal deformation and stress
structures with identical compositions
orderly arrangement of groups within the
properties depending on environment solution. Hence, liquid milk should, and does
show some type of arrangement.The mineral
‡ Viewing proteins in their native form
conditions
constituent and lactose are the only true
‡ Seeing difference between primary, crystalline constituents in dairy products that
can be analyzed by X-ray; nevertheless,
‡ Viewing how certain residues would
secondary and tertiary protein structure
interesting structural changes have been
interact and predict subsequent protein observed in butterfat, milk powder, casein and
cheese.
‡ Study of preferred orientation
folding

‡ Study of crystal anisotropy X-ray powder diffraction method is used for

powdery substances. Diffraction depends upon
Molecular Modeling Techniques the fact that in a fine powder, the particles are
Molecular modeling is a group of techniques arranged in an entirely heterogeneous manner.
that employ computer-generated images of Since reflection occurs from a definite angle,
chemical structures that show the relative there should be a sufficient number of particles
positioning of all the atoms present in the in the powder turned at just right angle to the
molecule being studied, and/or the simulated primary beam of monochromatic X-rays, to
dynamics of such molecules together with enable strong reflection from one set of
their ordering through spacetime. Such parallel planes; other particles turned at
techniques are of considerable help for another angle will produce reflection from
understanding many physicochemical another set of planes (the same set of planes in
many particles cooperating). Thus, a beam

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 Research and Reviews: Journal of Dairy Science and Technology
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passing through a powder specimen will fall produced α-form. Resolution was improved by
upon a perpendicular photographic film as a removal of liquid portion of fat by pressure
series of concentric rings, each of same filtration. In XRD pattern, a single strong band
intensity throughout and corresponding to one at 4.05 Å indicates α-form; two strong bands at
set of planes of spacing “d”. 4.2 Å and 3.8 Å indicate -form. HMF of milk
fat was obtained by crystallization of a 10%
Analysis of Milk Stones solution in acetone at 15 °C. [5]
X-ray diffraction technique has also been
applied for analysing the chemical Structure Elucidation of Milk Proteins
composition of milk stones. Since each Milk proteins play a range of roles covering
chemical compound gives a definite pattern on their wide range of nutritional and functional
a photographic film according to atomic properties. All of these behaviors relate to their
arrangement, X-rays can be used for structure and possible changes in structure of
qualitative chemical analysis as well as the component milk proteins during
structural analysis. processing. An understanding of the structure
of the milk proteins, and how those structures
X-Ray Analysis of Milk Powder can change under processing conditions, is
This technique has also been used in study of therefore an important enabling tool for the
milk powder. Most work has been confined to dairy processing industry.
determine the effect of different milk
powdering processes upon structural group Caseins
spacings within the milk proteins. Although, The single feature of casein structure that
structural changes within the milk protein due marks it out as different from other milk
to different types of processing equipment are proteins is its open and flexible environment-
not marked, there is a tendency for shrinkage dependent conformation, which has been
in unit spacing with an increase in heat termed rheomorphic. C-terminus of protein

residues with - angles in the region of

treatment. Hence, milk powders made by the comprises many stretches of consecutive
roller drying process have a tendency for a
smaller d unit spacing than do milk powders of polyproline II helix [6]. Two such helices
the spray types [4]. could, in principle, combine by hydrogen
bonding along the backbone chain, to form a
Differentiation of Sugar -sheet with a left handed twist [7], but this
Since each crystalline compound gives a does not occur extensively in caseins. This
definite pattern according to the atomic rheomorphic conformation gives the proteins
arrangement, the identification and the good foaming, emulsifying and gel-forming
differentiation of the common sugars (sucrose, properties and their remarkable stability to
dextrose and lactose) is made simple by X- heat. Caseins structure reveals that they do not
rays [4]. appear to fall under normal category of
globular proteins. To solve the structure of
Polymorphism in Milk Fat Shown by X-Ray non-globular casein molecules, one way is to
Diffraction and Infrared Spectroscopy form a complex with some other suitable
Occurrence of three polymorphic molecule, which is readily crystallizable and
modifications, viz., α, ’ and was studied by can be subjected to X-ray crystallographic
X-ray diffraction and infrared spectroscopy. analysis; alternately, caseins can be studied
Excellent agreement between the two methods using X-ray scattering technique [7].
was obtained. Slow cooling of milk fat
resulted in formation of ’ and -forms. Rapid Globular Milk Protein Structures
cooling of milk fat resulted in formation of α- Globular proteins tend to be those that are
form, which upon holding of sample at 5 °C, soluble and consequently relatively easy to
underwent transformation to ’ and -forms. purify. Resolving the structure of -
High-melting fraction (HMF) of milk fat lactoglobulin can help us understand the
obtained by crystallization from acetone processes of denaturation and aggregation,
existed in -form. Slow cooling of melted which lead, for example, to heat exchanger
HMF produced ’-form, and rapid cooling fouling during milk processing [8].

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Table 3: Three-Dimensional Structures of Molecules Relevant to Dairy Science [5].

Title Structure Experimental data Authors

Resolution [Å]: 2.30

†
†
R-Value: 0.195 (work) Groves, M. R.,

†
R-Free: n/a Dhanaraj, V.,

†
Chymosin Pitts, J. E.,
Space group: H32
†
complex with the Badasso, M.,

†
inhibitor cp- Unit Cell: Hoover, D.,

†
113972 [10] Nugent, P.,
Length [Å] Angles [°]
Blundell, T. L.
a = 132.78 α = 90.00
b = 132.78 = 90.00
c = 81.95 = 120.0

Resolution

†
[Å]: 1.15

†
Harata, K.,
R-Value: 0.122 (obs.)
†
Abe, Y.,
R-Free: 0.162 Muraki, M.
Human α- Space group: P 2 1 21 21
lactalbumin, low
temperature form Unit Cell:
[11] Length [Å] Angles [°]
a = 33.19 α = 90.00
b = 49.55 = 90.00
c = 64.20 = 90.00

Resolution

†
[Å]: 2.20

†
Chrysina, E. D.,
R-Value: 0.216 (obs.)
†
Brew, K.,
R-Free: 0.253 Acharya, K. R.
Crystal structure Space group: P 2 1 21 2
of bovine α-
lactalbumin Unit Cell:
[12] Length [Å] Angles [°]
a = 72.04 α = 90.00
b = 104.65 = 90.00
c = 117.42 = 90.00

Resolution
[Å]: 2.20 †
†
Chrysina, E. D.,

†
R-Value: 0.191 (obs.) Brew, K.,
R-Free: 0.248 Acharya, K. R.
Crystal structure
of apo-bovine α- Space Group: P 4 1 21 2
lactalbumin
[12] Unit Cell:
Length [Å] Angles [°]
a = 119.57 α = 90.00
b = 119.57 = 90.00
c = 152.74 = 90.00

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 Research and Reviews: Journal of Dairy Science and Technology
Volume 2, Issue 1, ISSN: 2319-3409
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†
Resolution [Å]: 2.40

†
R-Value: 0.210 (obs.) Kontopidis, G.,
Sawyer, L.
R-Free: 0.306
Bovine –
Space group: P 32 2 1
lactoglobulin
complexed with Unit Cell:
retinol, trigonal Length [Å] Angles [°]
lattice [13]
a = 53.58 α = 90.00
b = 53.58 = 90.00
c = 110.94 = 120.00

†
Resolution

†
[Å]: 2.50 Singh, A. K.,

†
Singh, N.,
Crystal structure R-Value: 0.189 (obs.)
†
Sharma, S.,
of chloride R-Free: 0.219
†
Kaur, P.,
saturated bovine
Space Group: P 21 Singh, T.P.
lactoperoxidase
at 2.5 A Unit Cell:
resolution shows
Length [Å] Angles [°]
multiple halide
binding sites a = 54.44 α = 90.00
[14] b = 80.56 = 102.77
c = 77.71 = 90.00

Resolution [Å]: 2.10

R-Value: 0.216 (obs.)
†
†
R-Free: 0.268 Vijayalakshmi, L.,

†
Krishna, R.,
Space Group: C 2 2 21
†
Sankaranarayanan, R.,
–lactoglobulin Unit Cell: Vijayan, M.
(native)
Length [Å] Angles [°]
[15]
a = 55.60 α = 90.00
b = 81.92 = 90.00
c = 66.99 = 90.00

Resolution
[Å]: 1.91 †
†
Mir, R.,

†
R-Value: 0.211 (obs.) Vikram, G.,

†
Crystal structure R-Free: 0.241 Singh, N.,

†
of c-lobe of Sinha, M.,
Space Group: P 21
†
bovine Sharma, S.,

†
lactoferrin with Unit Cell: Kaur, P.,
dextrin at 1.9 Å Singh, T. P.
resolution Length [Å] Angles [°]
[16] a = 61.81 α = 90.00
b = 50.13 = 107.10
c = 65.54 = 90.00

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 X-Ray Crystallography and Its Applications in Dairy Science Gandhi et al.
__________________________________________________________________________________________

Resolution

†
[Å]: 1.40

†
Abe, S.,
R-Value: 0.194 (obs.)
†
Koshiyama, T.,
R-Free: 0.212
†
Ohki, T.,

†
Crystal structure Space Group: P 4 3 21 2 Hikage, T.,

†
of hen egg white Watanabe, Y.,
lysozyme Unit Cell: Ueno, T.
[17] Length [Å] Angles [°]
a = 78.79 α = 90.00
b = 78.79 = 90.00
c = 36.96 = 90.00

Table 4: Some Milk Proteins for which There Are Coordinate Data in the Protein Data Bank [9].
Protein Source Method Coordinates Notes
Albumin Human serum X-ray lao6, lbj5 Several species
though coordinates
not available
Β-lactoglobulin Bovine X-ray. NMR lb0o, lbeb, lbso, Ovine, porcine,
[18] lbsq, lexs, lcj5 equine. Many more
coordinate sets
available
α-lactalbumin Buffalo X-ray. NMR l4v4, lalc, lb90, Several species.
[18] lhfx, lhmk, lhml Goat recombinant
protein.
Lactoferrin Human X-ray lbol, lblx, lbma, Equine and buffalo
lbiy, lcb6, llcf also
Galactosyl Bovine X-ray lfg5 Catalytic domain
transferase expressed in tissue
culture
Lactose synthase Bovine X-ray lj8w Atypical complex
Lipase Cow bile X-ray lakn, laql
Lysozyme Echidna milk X-ray ljng, lqqy Canine, also hen
egg white
IgG1 Human X-ray li4k, lcly Fc-fragment
complex
IgG2 Mouse X-ray Ljbg Fab fragment
IgA Mouse X-ray 2fbj Fab fragment
IgM Human X-ray Ladq Complex with IgG
fragment
2-Microglobulin Bovine X-ray Lbmg
Plasmin Human X-ray Lbml Catalytic domain
complex with
streptokinase
Xanthine oxidase Bovine milk X-ray Lfiq
Xanthine Bovine milk X-ray lfo4
dehyrogenase

CONCLUSIONS X-ray crystallography has been used for

Production of well-ordered crystals and analysis of liquid milk, milk powders, milk
generating X-rays of suitable energy and stones, polymorphism of milk fat and most
wavelength are the two primary requisites of widely and importantly in discovering the
X-ray crystallography. This technique has structure of most of the milk proteins and thus
widely been successfully used in elucidation of helping in correlating their structure with
detailed three-dimensional structures of possible functions.
biological molecules, especially proteins.

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 Research and Reviews: Journal of Dairy Science and Technology
Volume 2, Issue 1, ISSN: 2319-3409
__________________________________________________________________________________________

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