ROHIT SATWASE

M.Sc. Biotechnology
University of Mumbai.
INDEX
_Interference and diffraction
_X-rays
_Why X-rays?
_Crystal lattice and crystal system
_Braggs Equation
_Miller indices
_Steps involved in X-ray crystallography
_Crystallization
_Collecting X-ray diffraction data (x-ray sources, monochromator,
collimator, goniometer, camera,
detectors)
_Post data collection

INTERFERENCE AND DIFFRACTION

1) DIFFRACTION
Diffraction is the slight bending of light as it passes around the edge of an object.
(Dont imagine this bending as change in angle of travelling when seeing it side ways.
Instead imagine it to be a slight curving of the width of light wave. Yes wave does have some
width.
So this is what we mean when we say light bends around the edge of object.)

The amount of bending depends on the relative size of the wavelength of light to the size of
the opening.
If the opening is much larger than the light's wavelength, the bending will be almost
unnoticeable. However, if the two are closer in size or equal, the amount of bending is
considerable, and easily seen with the naked eye.
2) INTERFERENCE
The principle of superposition of waves states that,
when two (or more) propagating waves of same type arrive in a medium at the same point,
each wave produces its own displacement at that point independent of other. But, the total
(resultant) displacement at that point is equal to the vector sum of the displacements of the
individual waves.
If a crest of a wave meets a crest of another wave of the same frequency at the same point,
then the magnitude of the displacement is the sum of the individual magnitudes this is
constructive interference.
If a crest of one wave meets a trough of another wave then the magnitude of the
displacements is equal to the difference in the individual magnitudes this is known as
destructive interference.

Phase- measured in terms of . One crest or One trough =

Path- measured in terms of . One crest + One trough= dist. between 2 crests= dist. Between
2 troughs = = 2

Constructive interference occurs when :

(1) Phase difference between the waves is a 0 or multiple of or 2
OR
(2) Path difference = n ( where n = 0, 1,2,3..)
Whereas,
Destructive interference occurs when:
(1) Phase difference is an odd multiple of i.e n (where n=1,3,5..)
OR
(2) Path difference is odd multiple of /2 i.e n /2 (where n=1,3,5..)

In order to get steady interference pattern, following conditions are imp :

(1)Sources must be coherent i.e phase difference between them is either 0 or constant
(2)Two sources must be monochromatic i.e each source should emit wave of only one
wavelength
and
wave length of wave emitted from both should be same.
(3) Two sources must have waves of equal amplitude (height of crest and trough).
(4) The two sources should be narrow. If not, we will get many waves instead of one from
single source.
(5) Sources should be close to each other.
X-ray also show interference and diffraction and therefore these things are
applicable to x-rays !

X-RAYS
1) X-rays is a form of electromagnetic radiation. Most X-rays have a wavelength ranging from
0.01 to 10 nanometers.

2) X-rays are emitted by electrons.

Why x- rays?
Now one thing that comes to mind is why cant we visualizes molecules using visible light, why
we require x-rays for this purpose?
The answer to this question lies in their wavelength.
The wavelength of visible light is very large as compared to that of x-ray.
More smaller the structure to be visualised, more smaller the wavelength needed!
This can be illustrated with help of following example.
Suppose there is a car whose shape we want to know.
When we throw a ball at car, it will bounce of at particular angle depending on where it hits.
From the angle of bounce, the shape of car at that place can be deduced.
Now if we use larger ball, larger is the area of car hit and thus to cover the whole car we would

require less amount of balls. So the data accumulated would be very less. Hence, we wont get
that much details of the car shape.
On other hand if we use very small balls, less is the area of car hit everytime and thus to cover
the whole car we will need to do this repeatedly using many balls. So the data accumulated
would be more. Hence, we get much smaller details of the car.

Larger ball. Lesser details.

Smaller ball. Finer details.

Now imagine very very very large ball, larger even than size of car. Throwing such a huge ball
at relatively small car would be meaningless. The car will not at all influence the angle at
which the ball bounces back. Hence, we wont get any details about car shape.

Now compare the car with molecule, the very very huge ball with visible light and the small
balls with x-rays.
The size represents their wave length.
This explains why x-rays and not visible light.
Thus, x-rays, which have very small wave lengths, are used for determining the structure of

molecules.

CRYSTAL LATTICE AND CRYSTAL SYSTEMS

1) Crystal and crystal lattice
A crystal is a regular, repeating three-dimensional array of atoms or molecules or groups of
molecules.
If one imagines a point as representing each repeating unit, the array of points which
represents the crystal is called a lattice.
OR
Crystals are composed of three-dimensional patterns. These patterns consist of atoms or
groups of atoms in ordered and symmetrical arrangements which are repeated at regular
intervals keeping the same orientation to one another. By replacing each group of atoms by a
representative point a crystal lattice is obtained.

2) Bravais lattices
There are 14 different symmetrical patterns in which the points can be arranged in space
being consistent with the requirement that the patterns be repeated in all three dimensions
regularly to form the crystal.
These 14 patterns are called Bravais lattices.
These lattices are classified according to symmetry and space rotations into the seven crystal
systems.
3) Unit cell
The simplest repeating unit in a crystal is called a unit cell.
A unit cell is the most basic and least volume consuming repeating structure of any solid.
When the unit cell repeats itself, the network is called a lattice.
Opposite faces of a unit cell are parallel.
The edge of the unit cell connects equivalent points.

fig - The 14
Bravais unit cells

4) Crystal systems
-A triclinic unit cell has three unequal axes and angles.
Only one possible Bravais lattice.

-A monoclinic unit cell has three unequal axes, two of the angles as right angles, and no
limitation on the third one.
This condition can be obeyed both in the case where the lattice points are at the corners of the
unit cell, as well as when there is an extra lattice point on one of the faces. This is
conventionally taken to be the ab plane or the C face.
There are thus two possible Bravais lattices in this system.

- An orthorhombic unit cell has three unequal axes, and all three angles are right angles.
There are four possible Bravais lattices; with the lattice points at the corners; with an extra
lattice points on the ab plane; with an extra lattice point at the body centre of the unit cell; and
finally with extra lattice points on all six faces.

- A rhombohedral system has three equal sides and three equal angles.
only one possible Bravais lattice.

- A tetragonal system has two equal sides and all angles as right angles.
There are two possible Bravais lattices.

- A hexagonal unit cell.

It has two equal axes, and one angle as 120, the other two as right angles.

-A cubic system has equal sides and right angles.

There are three Bravais lattices.

Note - The Bravais lattices represent a complete set. No others are possible in a crystal.
5) Point Groups and Space Groups
Point groupCrystals can be classified in terms of the symmetry between their faces. In this case we
consider the crystal as a whole and ignore for the present the lattice structure underlying it.
Each crystal can be identified by the group of symmetry operations that pertain to it.
The symmetry operations can be of various types e.g- n fold rotation, and mirror planes,
centres of inversion etc.
Each group of symmetry operations that identify a crystal is called the symmetry point group
of that crystal.
a total of 32 point groups is possible for crystals and is divided among the seven crystal
systems discussed above.
Space groupThe three dimensional assembly of planes, axes and centres of symmetry is called a space
group.
Space group is the symmetry group of a configuration in space, usually in three dimensions.
There are 230 space groups, meaning 320 ways in which an object can be arranged in space to
form a crystal.

BRAGGS EQUATION
Diffraction of an x-ray beam, occurs when the light interacts with the electron cloud
surrounding the atoms of the crystalline solid.
Due to the periodic crystalline structure of a solid, it is possible to describe it as a series of
planes with an equal inter-planar distance d.
As an x-ray's beam hits the surface of the crystal at an angle say theta, some of the light will
be diffracted at that same angle theta away from the solid (see figure 2 below). The remainder
of the light will travel into the crystal and some of that light will interact with the second plane
of atoms. Some of the light will be diffracted at an angle theta, and the remainder will travel
deeper into the solid. This process will repeat for the many planes in the crystal.
The x-ray beams travel different path lengths before hitting the various planes of the crystal,
so after diffraction, the beams will interact constructively only if the path length difference is
equal to an integral multiple of wavelength!

See the figure above.

Consider the rays from outside to inside as ray 1, 2 and 3.
Take ray 1 and ray 2 for consideration. We can see that compared to ray 1, ray 2 has to travel
additional path length of BG before hitting the plane and length GF after diffraction.
Thus, the total difference in path lengths of the beam striking the first plane (i.e ray 1) and the
beam striking the second plane (i.e ray 2) is equal to BG + GF.
Now, for the two diffracted beams to constructively interfere the path length difference must
be equal to n !
i.e BG+GF = n
Say, ray 1 and ray 2 interfere constructively, therefore BG+GF= n

Now consider the ABG.

Here,
angle A =
we know
sin = opp side / hyp
= BG /AG
But,
therefore ,
therefore

AG = Interplanar distance = d
sin = BG / d
d sin = BG
.. (2)

similarly, in AFG.
we can prove
d sin = GF

.. (3)

. (1)

from (1) , (2) , (3)

2 d sin = n
This equation is known as Bragg's Law, named after W. H. Bragg and his son, W. L. Bragg; who
discovered this geometric relationship in 1912.
Bragg's Law relates the distance between two planes in a crystal and the angle of reflection to
the x-ray wavelength. The x-rays that are diffracted off the crystal have to be in-phase in order
to signal.

MILLER INDICES
>>>Miller indices form a notation system in crystallography for planes in crystal (Bravais)
lattices.
The Miller indices can be used to specify directions and planes in a crystal.
>>>The crystal lattice may be regarded as made up of set of parallel equidistant planes
passing through the lattice points which are known as lattice planes. In simple terms, the
planes passing through lattice points are called lattice planes.
For a given lattice, the lattice planes can be chosen in a different number of ways. See the
following fig.

The orientation of planes or faces in a crystal can be described in terms of their intercepts on
the three axes.
Miller introduced a system to designate a plane in a crystal.
He introduced a set of three numbers to specify a plane in a crystal.
This set of three numbers is known as Miller Indices of the concerned plane.
Thus the lattice planes are identified by a set of integers h, k, and . These are called Miller
indices.
When written (hk), round brackets without comma, it represents a plane.
There are also several related notations:
1) (h,k,l) ,round brackets with comma, represent a point.

2) The notation {hk}, with curly brackets denotes the set of all planes that are equivalent to
(hk) by the symmetry of the lattice.
3) The notation [hk], with square instead of round brackets, denotes a direction.
4) The notation hk, wih sharp brackets denotes the set of all directions.
Miller indices is defined as the reciprocals of the intercepts made by the plane on the three
axes.
>>>Procedure for determining Miller indices:
Step 1: Determine the intercepts of the plane along the axes X,Y and Z in terms of the lattice
constants a, b and c.
Step 2: Determine the reciprocals of these numbers.
Step 3: Find the least common multiple for the denominators and multiply each by this.
(You get three nos. Reduce to lowest terms if it can be)
Step 4: The result is written in parenthesis (i.e. in round brackets without comma).
This is called the `Miller Indices of the plane written in the form (h k l).
>>>Note- If a plane has negative intercept, the negative number is denoted by a bar above
the number.
e.g. 1) Plane ABC has intercepts of 2 units along X-axis, 3 units along Y-axis and 2 units along
Z-axis.

Find the miller indices of plane ABC .

Solution :
Step 1:The intercepts are 2,3 and 2 on the three axes.
Step 2:The reciprocals are 1/2, 1/3 and 1/2.
Step 3:The least common multiple of denominator is 6.
Multiplying each reciprocal by lcmd, we get, 3,2 and 3.
These are lowest terms and cannot be reduced further.
Step 4:Hence Miller indices for the plane ABC is (3 2 3)

>>>For the cubic crystal especially, the important features of Miller indices are,
- A plane which is parallel to any of the co-ordinate axes cuts it at infinity. So, it has an
intercept of infinity () for that axes.
Therefore the Miller index for that axis is zero (coz 1/ = 0).
Thus, for an intercept at infinity, the corresponding index is zero.
eg. 2) Find miller indices of plane parallel to y- and z-axis in unit cubic system.

Solution :
In the above plane, the intercept along X axis is 1 unit(since its unit cubic system).
The plane is parallel to Y and Z axes.
So, the intercepts along Y and Z axes are .
Now the intercepts are 1, ,.
Its reciprocal 1,0,0 .
Multiplying by lcmd we get same i.e 1,0,0.
It is already in lowest terms.
miller indices (100)
>>>All equally spaced parallel planes have same Miller indices i.e. The Miller indices do not
only define a particular plane but also a set of parallel planes.
eg.3)The planes whose intercepts are 1, 1,1; 2,2,2; -3,-3,-3 etc., are all represented by the
same set of Miller indices.
Prove it.
for 1,1,1 intercept > reciprocal 1,1,1 > mult. By lcmd 1,1,1 > already in reduced terms > (111)
for 2,2,2 intercept > reciprocal 1/2, 1/2, 1/2 > mult. By lcmd (i.e. 2)we get 1,1,1 > already in
reduced terms > (111)
for -3,-3,-3 intercept > reciprocal -1/3, -1/3, -1/3 > mult. By lcmd(i.e -3)we get 1,1,1 > already
in reduced terms > (111)
Thus, all these planes have (111) miller indices. It is a family/set of successive parallel planes
represented by {111}.

Set of Successive parallel planes in crystal have

have same Miller indices !!
eg.4)The (6,2,2) planes are the same as (3,1,1) planes.Prove.
6,2,2 > reciprocal 1/6,1/2,1/2 > mult. By lcmd (i.e 6) we get 1,3,3 > already in reduced terms
> (133)
3,1,1> reciprocal 1/3,1/1,1/1 > mult. By lcmd (i.e 3) we get 1,3,3 > already reduced terms >
(133)
>>>As said earlier, if a plane cuts an axis on the negative side of the origin, corresponding
index is negative.
It is represented by a bar, like ( 1

0 0) Miller indices indicates that the plane has an

intercept in the ve X axis.

>>>More solved eg.
e.g 5) A certain crystal has lattice parameters of 4.24, 10 and 3.66 on X, Y, Z axes

respectively.
Determine the Miller indices of a plane having intercepts of 2.12, 10 and 1.83 on the X, Y
and Z axes.
Lattice parameters are = 4.24, 10 and 3.66
The intercepts of the given plane = 2.12, 10 and 1.83
Lets consider crystal of given lattice parameters to be unit cell.
to get intercepts in terms of unit cell divide lattice parameters of each axes by the
intercepts made by plane
on respective axis.
Thus, the intercepts are, 0.5, 1 and 0.5.
Step 1:

The Intercepts are 1/2, 1 and 1/2.

Step 2:

The reciprocals are 2, 1 and 2.

Step 3:

The least common multiple for denominator is 2.

Step 4:

Multiplying the lcd by each reciprocal we get, 4, 2 and 4.

Step 5:

By writing them in parenthesis we get (4 2 4)

or we can also reduce to lowest terms after step 4 and get 2,1,2.
By writing them in parenthesis we get (2 1 2)

Therefore the Miller indices of the given plane is (4 2 4) or (2 1 2). Both are one and the same.

>>>Additional figures to get the concept

If sometimes we come across a plane that passes throug origin, ten it becomes problematic to
assign the intercepts because the plane dont intercept the axis but instead the edges of plane
are along the axis(or axes).
In such case, we take some another point of unit cell of crystal lattice as origin and do
calculations accordingly.

Basically what happens in crystallography is that the crystal is placed in one position one xrays are thrown on it. These x-rays get reflected from various successive parallel planes ( i.e.
family of planes having same miller indices).
The distance between these planes is constant since in crystal the molecules are arranged in
regular fashion.

As the distance is constant,all the x-rays reflected from these successive parallel planes
(family of planes with same miller indices) have same path difference.
As the distance between the molecules in crystal is very small, they act as natural three
dimentional grating (slits) for x-ray which have wavelength comparable to these slits. This
causes diffraction(bending through natural slits) of the x-rays as they emerge out of the
crystal.
The diffracted x-rays interefere and produce interference pattern.
These diffracted x-raysinterference patterns are recorded by detector.
Once this is done, the crystal is rotated slightly so that x-rays are incident on another set of
successive parallel planes, and the recordings are done.
Likewise, this thing is done for many sets of successive parallel planes and data is collected.
Using the data and various computational technologies, images are formed and super
imposed, again using computation, to yield the final structure of molecule.

STEPS INVOLVED IN X-RAY CRYSTALLOGRAPHY

The method of X-ray crystallography can be described very briefly as follows:
1) Crystallise the molecule,
2) Subject it to irradiation by a beam of X-rays and make a record of the 3-D diffraction
pattern.
3) Analysis of this data through computer calculations results in a model of the molecule,
which can then be refined against the observed X-ray data.
4) Finally one obtains the three dimensional coordinates, which describe the position in space
of each atom in the molecule. Such a refined model can be displayed and manipulated either
as plastic or wooden models, or on the computer graphics terminal.

CRYSTALLIZATION
If we want to determine the structure of a molecule, say a certain x protein, it is not possible
to work with a single molecule of xprotein and know its structure, because it would be very
difficult to bombard x-rays with that precision on such a small molecule. Instead if we have a
systematically arranged array of such molecules, i.e a crystal, we can easily bombard it with xrays, as its size is comparatively much bigger than that of single molecule, and carry out the
structure determination.

Hence, we use crystallized for of the molecules for structure determination using x-rays, the
technique being called x-ray crystallography.
1) Difference between biological(organic) and inorganic crystals
The chief difference:
In crystals of biological the repeating units are invariably molecules or groups of molecules,
while in the inorganic counterpart the repeating units are atoms or groups of atoms. This
implies that the forces of bonding between one repeating unit and another are much weaker in
the former, being van der Waals forces or hydrogen bonds. In the latter, on the other hand, the
crystals are built up from strong covalent or ionic bonds. Crystals of biological materials are
thus usually soft and brittle as compared to inorganic substances.
Another difference is the size:
Biological and organic molecules usually form very small crystals (approximately 1 cubic mm)
as compared to inorganic materials (about 1 cubic cm ).
Other difference:
Bio-crystals are usually grown from solution and may have a large solvent content. This is
especially true of crystals of macromolecules such as proteins and nucleic acids where the
solvent can take up as much as 80% of the crystal.

2) Factors affecting growth of crystal

Growth of bio-crystals is a multi-parameter process.
Table 7.1 gives a list of factors that could affect crystallisation.

3) Various methods of crystallization

-Inorganic crystals
Crystals of an inorganic substance can often be grown by preparing a hot, saturated solution of
the substance and then slowly cooling it. Polar organic compounds can sometimes be
crystallized by similar procedures or by slow precipitation from aqueous solutions by addition
of organic solvents.
-Organic crystals
However proteins and most other biological molecules are heat sensitive, they tend to
denature. Hence, various different techniques have been devised for their crystallization.
To grow crystals of any compound from solution, the molecules have to be brought to a

supersaturated state. When the solution returns to equilibrium, the substance will precipitate
out, hopefully as crystals, and not as an amorphous precipitate. This Supersaturation can be
achieved using following methods :
A) Slow evaporation is conceptually the simplest of techniques. It is chiefly used to
crystallise small organic molecules. The solution(protein + solvent) is placed in a small glass
beaker and sealed with an airtight cover, except for one or two small apertures through which
slow evaporation of the solvent takes place, bringing the solute to supersaturation and hence
to crystallisation.
eg- Purified protein is dissolved in an aqueous buffer containing a precipitant, such as
ammonium sulfate or polyethylene glycol, at a concentration, [precipitant], just below that
necessary to precipitate the protein. Then water is removed by controlled evaporation to raise
both [protein] and [precipitant], resulting in precipitation. Slow precipitation is more likely to
produce larger crystals, whereas rapid precipitation may produce many small crystals, or
worse, an amorphous solid.

[Note - Crystal formation occurs in two stages, nucleation, and growth.

Nucleation, the initial formation of molecular clusters from which crystals grow, requires
protein and/or precipitant concentrations higher than those optimal for slow precipitation.
In addition, nucleation conditions, if they persist, result in the formation of many nuclei, and as
a result, either an amorphous precipitate or many small crystals instead of a few larger ones.]
B) For biological macromolecules, especially, dialysis can be used to obtain crystals. The
solution of the molecule is separated from a large volume of the solvent by a semi-permeable
membrane which gives small molecules (ions, additives, buffer, etc.) the freedom to circulate
but prevents the movement of the macromolecule. The macromolecule solution is gradually
built up to supersaturation by changing the concentrations of the small molecules in the large
volume of the solvent. This has the effect of gradually changing the pH or the ionic strength or
any similar parameter.
C) One widely used crystallization technique is vapour diffusion, in which the
protein+precipitant solution is allowed to equilibrate in a closed container with a larger
aqueous reservoir containing precipitant such that the precipitant concentration is optimal for
producing crystals.
One of many examples of this technique is the hanging-drop method. In this Less than 25 L of

the solution of purified protein is mixed with an equal amount of the reservoir solution, giving
precipitant concentration about 50% of that required for protein crystallization. This solution
(protein+precipitant) is suspended as a droplet underneath a cover slip, which is sealed onto
the top of the reservoir with grease.
Because the precipitant is the major solute present, vapor diffusion (evaporation and
condensation) in this closed system results in net transfer of water from the drop
(protein+precipitant solution) to the reservoir, until the precipitant concentration is the same
in both solutions. Thus water is decreasing from drop slowly slowly , in creasing the conc. of
precipitant in it. This continues until equilibrium is attained.
When the system comes to equilibrium, net transfer of water ceases, and the protein solution
is maintained at constant precipitant concentration.
At this point, drop shrinkage has increased both [precipitant] and [protein], moving to the
nucleation region. As nuclei form, the protein concentration decreases and equilibrium gets
disturbed. Again there is transfer of water from drop to reservoir > drop shrinkage > moving
the conditions to the growth region. Thus, slowly saturation is occurring giving rise to crystal.
[Note- Seeding
Frequently the crystallographer obtains many small crystals instead of a few that are large
enough for diffraction measurements. These Small crystals of good quality can be used as
seeds to grow larger crystals. Alternatively seeds are usually obtained from previous less
successful crystallisation attempts, and these seed may be extremely tiny crystallites.
Seeding can be done in any of the methods .
Say, for e.g, seeding is done in hanging drop method. The experimental setup is the same as
before, except that each hanging droplet is seeded with a few small crystals. Seed crystals are
sometimes etched before use by brief soaking in buffer with precipitant concentration lower
than that of the mother liquor. This soak dissolves outer layers of the seed crystal, exposing
fresh surface on which crystallization can proceed.
Crystals may grow from seeds up to ten times faster than they grow a new. Sometimes it is
necessary to introduce seeds into the solution to induce the crystals to grow.]
Another variation in this type is the sitting drop technique in which the principle and procedure
is same except that in this technique, instead of hanging the drop from the airtight coverslip, it
is placed on some sort of support whose upper surface is well above the level of reservoir
solution.

D) Interface diffusion is used for small molecules as well as large biological macromolecules.
The precipitant solution is gently layered over the solution of the molecule.
Crystals usually grow at the interface.

Collecting X-ray diffraction data

The goal of data collection is to determine the indices and record the intensities of as many
reflections as possible, as rapidly and efficiently as possible.
One cause for urgency is that crystals, especially those of macromolecules, deteriorate in the
beam because X-rays generate heat and reactive free radicals in the crystal. Thus the
crystallographer would like to capture as many reflections as possible during every moment of
irradiation.
Protein crystals that produce measurable reflections from interplanar spacings down to about 3
or less are usually suitable for structure determination.
The major instruments employed in data collection include
1) The X-ray source which produce an intense, narrow beam of radiation.
2) Monochromator ( an optical device that transmits a mechanically selectable narrow band of
wavelengths of light or
other radiation chosen from a wider range of wavelengths available at the input)
and
Collimator (a device that narrows a beam of particles or waves)
3) Goniometer, an instrument that allows an object to be rotated to a precise angular position.
3) Cameras, which control the orientation of the crystal in the X-ray beam, and thus direct
reflections having known indices to a detectors.
5) Detectors, which allow quantitative measurement of reflection intensities.

We will see these instruments/devices in details.

A) X-ray sources
X-rays are electromagnetic radiation of wavelengths 0.1100 .
X-rays in the useful range for crystallography can be produced by bombarding a metal target
(most commonly copper, molybdenum, or chromium) with electrons produced by a heated
filament and accelerated by an electric field.
The high-energy electron collides with and displaces an electron from a low-lying orbital in a
target metal atom.
Then an electron from higher orbital drops into the resulting vacancy.
We know electron in higher orbital has more energy than electron in lower orbital. Therefore,
when electron jumps from outer to inner orbital, it emits its excess energy as an X-ray photon.
Hence, the metal in the target exhibits narrow characteristic lines (specific wavelengths) of
emission resulting from the characteristic energy-level spacing of that element.
The wavelengths of emission lines are longer for elements of lower atomic number Z.
e.g. Electronic shells of atoms are designated, starting from the lowest level, as K, L, M, . . ..
Electrons dropping from the L shell of copper (Z = 29) to replace displaced K electrons (L K
or K transition) emit X-rays of = 1.54 . The M K transition produces a nearby emission
band (K) at 1.39 .
For molybdenum (Z = 42), (K) = 0.71 , and (K) = 0.63 .
Elements like copper and molybdenum make good X-ray sources if the weaker K radiation
can be removed.
There are three common X-ray sources:
- X-ray tubes (actually a cathode ray tube sort of like a television tube),
- rotating anode tubes,
and
- particle storage rings, which produce synchrotron radiation in the X-ray region.
(1) x-ray tubes

In the X-ray tube, electrons from a hot filament (cathode) are accelerated by electrically
charged plates and collide with a water-cooled anode made of the target metal.
X-rays are produced at low angles from the anode, and emerge from the tube through windows
of beryllium.
Output from X-ray tubes is limited by the amount of heat that can be dissipated from the
anode by circulating water.
(2) Rotating anode tubes
Higher X-ray output can be obtained from rotating anode sources, in which the target is a
rapidly rotating metal disk.
This arrangement improves heat dissipation by spreading the powerful electron bombardment
over a much larger piece of metal.
Rotating anode sources are more than ten times as powerful as tubes with fixed anodes.
(3) Particle storage rings
Particle storage rings, which are associated with the particle accelerators used by physicists to
study subatomic particles, are the most powerful X-ray sources.
In these giant rings, electrons or positrons circulate at velocities near the speed of light, driven
by energy from radio-frequency transmitters and maintained in circular motion by powerful
magnets.
A charged body like an electron emits energy (synchrotron radiation) when forced into curved
motion, and in accelerators, the energy is emitted as X-rays.
Accessory devices called wigglers cause additional bending of the beam, thus increasing the
intensity of radiation.
Systems of focusing mirrors and monochromators tangential to the storage ring provide
powerful monochromatic X-rays at selectable wavelengths.
Although synchrotron sources are available only at storage rings and require the
crystallographer to collect data away from the usual site of work, there are many advantages
that compensate for the inconvenience. For e.g. X-ray data that requires several hours of
exposure to a rotating anode source can often be obtained in seconds or minutes at a
synchrotron source like NSLS. In a day or two at a synchrotron source, a crystallographer can
collect data that might take weeks to acquire with conventional sources.

Fig 4.23 - A particle-storage ring designed expressly for producing X-rays, part of the National
Synchrotron Light Source (NSLS) at Brookhaven National Laboratory on Long Island in New
York, is shown in Fig. 4.23. In the interior floor plan (Fig. 4.18b), paths of particle-storage rings
are shown as dark circles. The largest of the three rings in the building is the X-ray ring, which
is 54 meters (177 feet) in diameter. The dark lines tangent to rings represent beam lines for
work in experimental stations (green blocks). NSLS began operations in 1984.
Crystallographers can apply to NSLS and other synchrotron sources for grants of time for data
collection.
B) Monochromtor
The monochromators employed usually are crystals of topaz, sodium chloride, quartz or heavy
metal fatty acids(e.g. lead palmitate or strontium behenate).
A monochromatic radiation is obtained by reflecting x-rays from crystal placed at a specific
angle.
For shorter wavelengths, crystals having small d-spacing are used. Crystals having larger d-

spacing yield x rays of higher wavelength.

C) Collimator
The Collimator is a narrow metal tube that selects and reflects the X-rays into parallel paths,
producing a narrow beam.

D) Goniometer
The crystal is mounted for measurements so that it may be held in the X-ray beam and
rotated.
There are several methods of mounting.
In the past, crystals were loaded into glass capillaries with the crystallization solution (the
mother liquor).
Nowadays, crystals of small molecules are typically attached with oil or glue to a glass fiber or
a loop, which is made of nylon or plastic and attached to a solid rod. Protein crystals are
scooped up by a loop, then flash-frozen with liquid nitrogen. (Note- This freezing reduces the
radiation damage of the X-rays. The crystals are generally pre-soaked in a cryoprotectant
solution before freezing; untreated protein crystals often crack if flash-frozen)
The capillary or loop is mounted on a goniometer, which allows it to be positioned accurately
within the X-ray beam and rotated.
The most common type of goniometer is the "kappa goniometer", which offers three angles of
rotation: the angle, which rotates about an axis perpendicular to the beam; the angle,
about an axis at ~50 to the axis; and, finally, the angle about the loop/capillary axis.
When the angle is zero, the and axes are aligned. The rotation allows for convenient
mounting of the crystal, since the arm in which the crystal is mounted may be swung out
towards the crystallographer. The oscillations carried out during data collection (mentioned
below) involve the axis only.
An older type of goniometer is the four-circle goniometer, and its relatives such as the sixcircle goniometer.

E) Camera
Since both the crystal and the beam are often very small, the crystal must be centered within
the beam to within ~25 micrometer accuracy, which is aided by a camera focused on the
crystal.
Thus, between the X-ray source and the detector lies a mounted crystal, in the grip of an X-ray
camera.

Thus, over all in the setup so formed, the x-ray source emits x-rays which emerge through the
monochromator and collimator as narrow monochromatic waves of x-ray. These x-rays fall on
the crystal mounted in loop placed on goniometer. The camera helps to centre the crystal in
the x-rays. When the x-rays fall on the crystal, they are reflected from one parallel set of
planes denoted by millers indices. When these reflected x-rays emerge out of crystals from
various planes of parallel set, they tend to diffract as the size of the molecule spacing in
crystal is very small. These diffracted x-ray interfere and cause interference pattern. With the
help of goniometer we rotate the crystal at various angles so that x-rays are diffracted from
different sets of parallel planes of the crystal and the diffraction pattern is recorded from many
such sets. These diffracted x-ray patterns are collected by detectors which are explained in
following section.
F) Detectors

POST DATA COLLECTION

The outcome of the data collection process is a list of angles, indicating the orientation of the
crystal and the detector and the intensity of the reflection from the oriented lattice plane at
this position. Instead of the angles, it is usual to identify the set of planes that give rise to each
diffracted spot by the Miller indices hkl and associate with each value of hkl the appropriate
value of the intensity.
From the list of intensities, the amplitudes of each reflected wave is extracted by the simple
process of taking the square root, since the measured intensity of a wave is the square of its
amplitude.
These amplitudes, represented as Fhkl are now used in the analysis of the structure of the
molecule that makes up the crystal.
In order to obtain structure of molecule, we do something called as Fourier transform of
observed amplitudes Fhkl.
But, before Fourier transform, it is necessary to know the phases of these waves. The phases
cannot be measured directly and the phases have to be determined by other indirect means.
Some of these methods are described below:
- 1 Heavy atom or multiple isomorphous replacement (MIR) method
- 2 Direct methods
- 3 Molecular replacement

- 4 The anomalous scattering technique

Once the approximate value of the phases are determined the next step is to refine them.
This usually more conveniently done by performing the Fourier transform and refining the
approximate positions of the atoms obtained by including other known data such as
stereochemistry.
Some methods used are :
- 1 Least squares method
- 2 Constrained-restrained refinement
- 3 Computer graphics in refinement
At the end of the refinement process we obtain a set of coordinates for each of the atoms of
the molecule and another number (or set of numbers) representing the thermal vibrations of
the atom.These can be subjected to further geometrical analyses to understand the chemistry
or biology of the molecule.