ation

1adation wWWWM
w,
AwWVM,
scatlered

V
nelix a
161

scattered waves
are in phase
at this angle
distance repeat
C
between
atoms
X-Ray Diffractic
11
path difference equals
one wavelength at this angle pitch, P

regularly

row
of
s
opna
ec e da t o m s o r

molecules

a row of
simple structure
-

sin
from a very
tion
D i f t r a c t

oms or moiecules.

allowed to pass through a

Fig. 2. A simple hellcal molecule.
aradiation is
hen ructure,
gular, repeating s t r u c t
fraction is observe
by repeating the
In 1912 Knipping and Frinedrich in Laue's laboras scattered
The rule relating the
that radiation periodic spacings in object
shows reintorcement of
the first X-ray diftraction experiment with a crystal Thmis me in he the structure
structure
and diffraction pattern is simple: short spacings in
directions
showed at the same time that Ä-rays behave as waves elements
waves in c e r t a i n specific the periodic structures correspond to large spacings
tscattered directions. A
crystals display a high degree of order in arranging theirand a of the w a v e s in other
in the diffraction pattern, and vice versa. In addition.
alons weakening is given in Figure 1, which shows
ions or molecules. The crystal acts as a three-dimensional nle example Scattered Irom a r o w of equally by determining the relative intensities of different
and diffracts X rays just as visible light is diffracted by an gralung mple
we can tell how matter is distributed within
optig niation being directions will the
spots,
in certain
grating. Since 1912, X-ray crystallography has had an enom enormouECed atoms. Only
each repeat of the structure.
d n in phase and thererore
impact on chemistry and biology. At first, great triumphs edwaves be
obtained in the field of interfere with (reinforce) one another. Fiber Diffraction
inorganiC chemistry.The structures of simpe sructively
ionic compoundsand of complex structures. like the silicates, bealother directions they
will be another.
Outo Thus, consider first the diffraction from helical
with one
We
understood, and ionic radii could be measured with high destructively interfere
accuracy difraction molecules, aligned approximately parallel
to the axis
Unlike inorganic compounds that are mainly ionic, organic compound pattern is generated. For the diftracuon
molecule, like the one
of a stretched fiber. A helical
be sharp.
by onguikem 1s
consist of molecules. The intensity of the beams diffracted to
of the radiation used be somewnat
it essentia shown in Figure 2, 1s
characterized by certain

Compounds 1s relatively low and, in the beginning, progress waawelength between tne parameters:
SIOw in determining their structures. However, the introduction oforter than the regular spacing is why X-rays are
The repeat (c) of the
helix is the distance parallel
mproved hardware and Fourier analysis solved the problem, adeents of the structure. This which I the structure exactly repeats
to the axis in number (n)
now the
X-ray structure determination of a small or medium-sz in studying molecules, since x-rays typicallya itself. The repeat
contains some integral

a Wavelength of only a few tenths of 2, 4. =

n
Organic molecule is an easy job, at least wher good quality cryou residues. In Figure
of polymer
is the distance parallel
are examined. Cet. it the regular spacing in the object being The pitch (p) of
the helix
makes one turn.
Ony a few decades ago, virtually nothing was known about u arge (as in a windoW screen), we can
axis in which the helix
to the helix residues per turn
the phenomenon with visible number of
Lhree-dimensional structures of nucleic acids, proteins, EXactly same
If there is
an integral
equal.
polysaccharides. Today, largely as a result of the technigucof x- which has a wavelength thousands of times (as here),
the pitch
and repeat
is the
are
distance parallel
diffraction, many of these molecules are understood at a level o than x-rays. We will find that a point source, The rise (h)
of the helix
residue to the
next,

of one
that would have rough a window screen. gives a rectangular axis from
the level
surprised the biochemists of 1950. 1he HChon to the
cO Caled and it is
possible
give only a brief introu
to
pattern of spots.
Oescribing what is measured and what can be ined. BC43
(BC-43)
 X-Ray DrCHon between many chemical 163
angles
162
direction of
fiber axis
knguhsand
d
the

m u s t
also
be inspected to see

approach closer than

their vander
nroach
m o d e l

model, the intensities of

The
fiber:a O m s

uch a
wo
dii. From
p r e d i c t e d .
ted. These predictions
no be
can
intensities, and the
spots
c o m p a r e a W I t h t hee
observed
various
best fit is obtained. The
elisreadjusted until a
inversely structure of DNA was
proportional to the
determination o f the

rise to helix one c a n

see from Figure
As
this way.
v are not as neat as
diffraction patterns
inversely
in
because of incomplete
proportional to ample, mainly
repeat distance m o l e c u l e s .

jidealized

the
of
gnent
Crystal Ditfraction

such as those formed

crystals
molecular molecules like tRNA, and
sudy
oligonucleotides, rather different
faces a
one
proteins, of helical fibers and
the study
em from different way. A schematic
quite
in a
1s shown in Figure 4.
a crystal
oceeds
of such unit cell, which may Fig. 4. Scheme drwaing of a molecular crystal. |
drawing is noW the
erepeating unit molecules. The unit cell
or more
two,
block of information can be extracted than from a fiber
ntain one, of as the basic building
RY be thought of the unit cell in three diffraction pattem, because the coTesponding
TiDer
incoming crvstal. Repetition a r o w s on the figure) creates molecules in each unit cell of the crystalare of the

dmensions (marked by oriented in the way. In

radiation same
two-dimensional analog same shape and are

whole crystal. A simple fiber diffraction, the helical molecules may

all have
pattern in
unit cell is the repeating the same direction, but
the crystal random a wallpaper their long axes pointed in
No matter how about these axes. This
Fig. 3. Diffraction from fibrers (A) A fiber in an x-ray beam. (B) The diffraction pattem. allpaper. stare at it long enough, they are rotated randomly be
seem, if you of arrangement can
putern may fills difference in exactness

can always
find a unit that, by repetition, the sharpness of the
with a helical structure. The spots all lie on
jou diffraction, passing appreciated by comparing with
as in fiber shown in Figure 5
so h dn. If we think of a spiral staircase as an
perpendicular to the fiber axis; these are called la he entire wall. Just a molecular crystal produces crystal diffraction pattern
4.
example of a helix, the rise is the height of each step beam through in Figure
lines. The spacing between these lines is
-ay
adifraction pattern.
the fiber pattern depicted from a
and the pitch is the distance from where one is diffraction pattern
proportional to the repeat of the helix, c, whih obtained After obtaining the the
standing to the corresponding spot directly overhead. The pattern shown in Figure 5 was
the experimenter
measures

the molecular crystal, If the

this case the
Suppose we wish to investigate a polymer with the pitch. Note
equals that croSma crystal of a small DNA. Again. spacing intensities of a large
number of the spots.
it is possible
repeats itself on every fourth layer lne.
the repeating
the helical structure shown in Figure 2. A fiber is
the spots allows us to determine Case molecule being studied is a small one,
with fiber
pulled from a concentrated solution of the polymer. repetition pattern tells us that there ac ances in the periodic structure- in this in much the same
manner as

expected
the niset e labelled to proceed and
Stretching the fiber further will produce approximate residues per turn in the helix. Thus, z dimensions of the unit cell is guessed
alignment of the long helical molecules with the helix is c/4. This is the kind of evidence tna
1and patterns.A
structure
with the
0and c in Figure 4. But the important are
calculated and compared
refined until
fiber axis. The fiber is then Watson and Crick that B-DNA was a neu how intensities
is
placed in an x-ray crystal diffraction studies is Just
structure
in The
beam, and a photographic film is On observed
intensities.
are correctly
positioned behind 10 residues per turn. atoms within each unit cell, tor of all spots
it, as shown in Figure 3a. The arranged relative
intensities
won't work
this the
diffraction pattern, The information above is given direcuy a rangement describes the molecule. Again, However,
such a procedure
there is
which consists of spots or short all of
the predicted. the tRNA,
that in Figure 3b. It can
arcs, will look like pattern. To find out exactly how a lormation is contain in the relative intensities molecule as
complex
as
structure.
be read as follows: n each residue are arranged in each repe the diffraction spots in a pattern like
that shown with a
to guess
such a

According to the mathematics of difraction ure , But in crystal diffraction, more Kact Simply
no way
a helix
always gives rise to this kind of
theory, detailed analysis is necessary. Usualy
nd r
shaped pattern. Theretore, we know cross- made using the correct repeat, pite
we are
000 appro.

dealing making is simplified because we know

(BC-43)
 Ray Dire
164
165

G12

c3
0.20 nm resolution
0.15 nm resolution
0.12 nm resolution
Fig. 7. Effect of increased resolution on molecular details observed by A-ray diffraction.

factors and, from then the

structure

lculated e n s i t y distribution. These calculatior

Fig. 6. Part of an electron density map de computer,
DNA crystal diffraction pattern. derived elcciro

uSually
done on
a large
will first carry
re tthe
he investigator
cases,
cases,

by a molecular crystal
most
with :a relatively small number
Diiraction pattern produced Usually multiple isomorphous repla in analysis
Fig. 5. his will give a low-resolution
of a small DNA. necessary to determine the phases ofNhog
This procedure more spots will be
pots
is going well,
from spot
intensities factors. If all refined to give highe
gher
Why proceed directly
not
Given structure factors for all of the
icture.
the
calculations

some of the ired and

difficulty is that it is now possible
crystals,
the structure? The positions of spots
best
to
is investigator can calculate the al With the
the spot intensities 0.10 nm. This
information contained in al s l u t i o n . about
oI
in the unit cell. What is actually resolutions

hidden. To greatly simplify

a complex problem,
we
calculated obtain
is sufficient
to 1dentify individual groups
that the electron density distribution (Fig. 6, solhution interact
may say that it is as if the quantities but atoms and to
show how they
amounts to the same thing, for some
experimenter needed in order to
deduce the structure regions of nd even detail in the phenolic ring of
another. The
(which are called structure factors) were the square
electron density are are lith one
where the atoms
revealed at different
resolutions
has a value particular view shown in Figure 6, we are l wotein side chain
roots of the intensities. *If the intensity

of, say, 25. the investigator knows that the

number at a two-dimensional "slice" through the shownin Figure three-dimensional structures
dimensional electron density distribution. Most of the detailed
needed is +5 or 5. But which? This sort of
-

macromolecules shown in this book have

appropriate to review the sten iological
quandary is the essence of the phase problem, SoW diffraction studies.of
which prevented progress in large-molecule to determine the three-dimeniben determined by X-ray
must be taken present, hundreds of such structures are
difirystals. At
crystallography for many years. One way of solving
Structureof a macromolecule from crystal an enormous
the problem was discovered in the early 1950s. studies: nown. This knowledge represents but the results
of labor in many laboratories,
Suppose a heavy metal atom can be introduced (1) Obtain satisfactory crystals. This step s d amount function at a
molecular
into some point in the molecule in such a way that the hardest part of the procedure, for alow us to understand macro short
el that would have been unbelievable only
a
the molecule and crystal are otherwise unchanged. crystals must be of good quality and at lea
This process is called an isomorphous few tenths of a millimeter in mini E Ago.
replacement.
Now suppose the heavy metal contributes a value dimension. Crystals that are too small wil
of +2 the structure factor for the
Instrument Fig. 8. X-ray
diffraction spectrophometer.
to
spot we were give sharp diffraction patterns. Geting m
just discussing. If the original value was +5, its
molecules to crystallize well is still
moe ams
by crystals of small compounds
diffracted
and is equipped
a crystal
new value is +7 and its
square is 49. If the original
value was -5, the value now becomes -3 art than a science.
detected and measured
Lsually with one or
more axes
to orient

which
the
hundreds or
thousands

and its (2) Record the diffraction pattern from the rctometer. Using this instrument, the crystal can with an area
detector on
registered nearly
square is 9. The investigator takes a diffraction of the ed around three or four axes and can reacn beams
are
plate
and measure the intensities of many
diffracted
of of the imaging
photograph of the crystal with the heavy metal way to make
isomom every orientation. The diffracted beams are simultaneously.
A popular type material. After
inserted. If the new (3) Find some phosphorescent

for this spot, the

crystal has an intensity I of 9 replacements in the molecule. Usuaiuy lied with a photon counter. Because the beams is covered
with
collected on the inmaging
original structure factor must have Lred one after the other, this process is slow registration,
the
information

and read with a

been 5 not +5. more replacements are required. not very a laser

this example
Although an
oversimplification, (4) Repeat steps 1 and 2 for eacn
ISOmop
suitable for protein crystals wnerc plate is
replaced by
gives the essence of the method. Usands of beams must be measured.
Protein
photomultiplier.

derivative. has
4ographers prefer an instrumen that
 166
X-Ray Diffraction
Applications for small organic compounds: the
method o
metho
Protein
crystallography isomorphous replacement in which
h heavw
X-ray crystallography.
is the youngest branch of
is attached to the protein structure.
aa
It
heavy atom
It started in 1934 when was Pemd
Bernal took the first achievement to show
that the method could ruti
X-ray diffraction picture of be used for al
a
protein crystal, first a crystal of proteins and that the heavy
pepsin,
larger change in intensity atoms
soon
followed by a crystal of insulin. cause a much
observations showed that even These initially expected. Because of the rather than
these large primitive
molecules are nicely ordered in their crystals and instrumentation, progress was slow at first.
that their structures the With
might be solved. This was not introduction of more
an easy
problem, however, and it took instrumentation and structures
sophisticated
years to find a solution. twenty with ever
are being published
Surprisingly, it could be increasing speed. They are collected in
done by a
technique already successfully applied the Protein Data
Bank, Brookhaven National
Laboratory, Upton, NY 11973-5000, USA.
Important
1.
Questions
Describe in details the process of
2. X-ray diffraction with special reference to its
Differentiate between the fiber diffraction and crystal diffraction
with suitable applications.
examples.