2. Experimental methods Computational methods
X-ray crystallography Homology Modeling
Nuclear Magnetic Resonance (NMR) Fold Recognition
Electron Microscopy (EM) Free Modeling
Hybrid
*Others
Methods used for obtaining three-dimensional structures of proteins
*Other methods:
Spectrophotometric methods such as circular dichroism (CD) provide details on the helical
content of proteins. UV-visible absorbance spectrophotometry assist in identifying metal ions,
aromatic groups or co-factors attached to proteins, whilst fluorescence methods indicate local
environment of tryptophan side chains.
3. The impact of structural methods on descriptions of protein function includes understanding the
mechanism of oxygen binding and allosteric activity in haemoglobin as well as catalytic activity of
enzymes.
PDB statistics
Experimental method Proteins Nucleic acids Protein/NA complex Other Total
X-ray 77139 1481 4059 3 82682
NMR 8829 1044 193 7 10073
Electron microscopy 466 45 128 0 639
Hybrid 51 3 2 1 57
Other 150 4 6 13 173
Total 86635 2577 4388 24 93624
Over 88% (82,682) of all experimentally derived structures are the result of crystallographic studies, 10%
(10073) solved using NMR spectroscopy and 1% (639) by cryoelectron microscopy (cryo-EM).
4. What is the common factor in all these methods?
5. The electromagnetic spectrum extends over a wide range of frequencies (or wavelengths) and
includes radio waves, microwaves, the infrared region, the familiar ultraviolet and visible
regions of the spectrum, eventually reaching very short wavelength or high frequency X-rays.
The use of electromagnetic radiation
6. The energy (E) associated with radiation is defined by Planck’s law
E = hν where ν = c/λ
Where c is the velocity of light (3 x 108 ms-1) and h, Planck’s constant, has magnitude of 6.6 x
10-34 Js and ν is the frequency of the radiation.
The ultraviolet (UV) and visible regions of the electromagnetic spectrum are of higher energy
and probe changes in electronic structure through transitions occurring to electrons in the
outer shells of atoms. Fluorescence and absorbance methods are widely used in protein
biochemistry and are based on these transitions.
Finally X-rays are used to probe changes to the inner electron shells of atoms. These
techniques require high energies to knock inner electrons from their shells and this is reflected
in the frequency of such transition (~1018 Hz). The X-rays have very short wavelengths of
~0.15 x 10-9 m or less.
7. All branches of spectroscopy involve either absorption or emission of radiation and are
governed by a fundamental equation
ΔE = E2 – E1 = hν
where E2 and E1 are the energies of the two quantized states involved in the transition. Most
branches of spectroscopy involve the absorption of radiation with the elevation of the atom or
molecule from a ground state to one or more excited states.
9. Technique Frequency range (Hz) Measurement
NMR 0.6 – 60 x 107 Nucleus’ magnetic field
ESR 1 - 30 x 109 Electron’s magnetic field
Microwave 0.1 - 60 x 1010 Molecular rotation
Infrared 0.6 - 400 x 1012 Bond vibration and bending
Ultraviolet/visible 7.5 – 300 x 1014 Outer core electron transitions
Mossbauer 3 – 300 x 1016 Inner core electron transitions
X-ray 1.5 – 15 x 1018 Inner core electron transitions
The frequency range and atomic parameters central to physical techniques used
to study protein structure
11. Of perhaps greater significance was the research of Lawrence Bragg, working with his father
William Bragg, who interpreted the patterns of spots obtained on photographic plates located
close to crystals exposed to X-rays.
Bragg realized ‘focusing effects’ arise if X-rays are reflected by series of atomic planes and he
formulated a direct relationship between the crystal structure and its diffraction pattern that is
now called Bragg’s law.
Bragg recognized that sets of parallel lattice planes would ‘select’ from incident radiation
those wavelengths corresponding to integral multiples of this wavelength. Peaks of intensity
for the scattered X-rays are observed when the angle of incidence is equal to the angle of
scattering and the path length difference is equal to an integer number of wavelengths.
The path difference
nλ = 2d sinθ
12. The crystalline state
What are the states of matter?
1. Gases: fill entire volume available to them, change their volume in response to pressure,
have low density and free flow.
2. Liquids: occupy fixed volume at a temperature, assume the shape of the container,
slightly compressible, density is little higher than gases.
3. Solids: have fixed size and fixed shape, high density, virtually incompressible.
What are molecular structures of gases, liquids and solids?
13. Conductive properties:
Graphite shows different electrical values on different sides of directions.
This variation of physical property with direction is referred as anisotropy
and graphite is said to be anisotropic with respect to electrical
conductivity.
Anisotropy
Mechanical properties:
Solid mica can be cleaved very easily into fine layers. However, it not
easily cleaved if tried from other side than parallel to the nature layer
structure. Thus, mica shows anisotropy in its mechanical strength with
respect to cleavage.
What is anisotropy?
Thermal properties:
Some solids shows different expansion in different direction on heating. Hence thermal
expansion shows anisotropy.
14. Optical properties:
When light beam incident on calcium carbonate
(calcite), then there are two refracted beams, known as
birefringence. Moreover, the two beams are polarized in
different directions and is it is found that the velocity of
light in the material varies with the direction of
propagation of light within the mineral. This is an
example of optical anisotropy in the solid state.
Magnetic properties:
Ferromagnetic materials may be magnetized more easily in some directions than in others,
showing that these materials exhibit magnetic anisotropy.
Electrical properties:
For many solids the magnitude of the dielectric constant varies with direction. (The dielectric
constant is related to the strength of an electric field with the solid and is determined by the
dipole moment of the molecules in the material).
15. Significance of order
Given that anisotropy is a fundamental characteristic of many solids:
Can we make any deductions which are relevant to our understanding of the structure of
solids? Or
What feature of the structure of the solid state will give rise to anisotropy?
16. Methane is a highly symmetric molecule, both spatially (tetrahedron) and structurally (all H).
Chlorobenzene is different in one respect, chlorine atom is more electronegative than the
benzene groups, chlorobenzene has a dipole moment directed along the benzene ring C-Cl
bond. The direction parallel to this dipole moment defines a special direction in space, a
direction determined by the structure of the chlorobenzene molecule and a direction which
defines anisotropy on a molecule scale.
Thus, individual molecules can enable them to have particular directional properties and
anisotropy can be explained on a molecular scale as being fundamentally due to molecule
structure.
17. Let us now turn to a multi-molecular aggregate of molecules as in a solid. Let us consider two
ways of packing chlorobenzene molecules together.
Which of these structures is anisotropic?
A random array, no net dipole moment. A regular array, a net dipole moment exists.
Which of these arrangements will have a net dipole moment?
18. Thus, we can see that it is the ordering which is the clue to the significance of anisotropy.
What is the difference between the array which gives rise to anisotropic effects and the
array which is isotropic?
Since the physical properties of the solid state necessarily reflect the properties of very large
numbers of individual molecules, it is only when these molecules are arranged in a define,
well-defined, ordered array that any directional properties may become apparent. If the
arrangement is random, then any directional property of the component molecules will be
average to zero o account of the random irregular orientation and position of one molecule
with respect to the next.
Anisotropy is possible only when the molecules are arranged with regularity and order.
19. Are all solids which are ordered, anisotropic?
It is not always true that all well-ordered arrays necessarily exhibit anisotropy.
However, it is true that all anisotropic materials necessarily have an ordered structure.
20. Solid: The molecules are
closely spaced with strong
intermolecular forces e.g. in
a well-ordered, long-range
three-dimensional array.
Liquid: The molecules are
quite close and although
each molecule has about the
same number of nearest
neighbors, there is no long-
range order.
Gas: The molecules
are far part and
independent of one
another. There is no
ordering at all.
Exercise: are all gases isotropic?
21. Crystals
The existence of anisotropy is possible if and only if the molecules in a material are ordered in
some systematic manner.
We say that solids are more ordered than liquids. But how do we measure the
orderedness?
Any assembly which maintains its order over a greater distance is more ordered than one
which is ordered over only a comparatively short distance. A significant measure of distance
for a molecular system is the average intermolecular spacing . Thus we may say that an
ordered solid preserves the ordering of its structure over many more intermolecular spacing
than does a liquid. For solids, e.g. 106 intermolecular spacing.
Thus, ordered solid state is characterized by a long-range order which extends over literally
millions of molecules, so that the environment of any one molecule is identical to that of any
other molecule.
22. Solids which possess this long-range, three-dimensional ordering are known as crystals. A
crystal may thus be defined as a solid which possesses long-range, three-dimensional
molecular order.
A direct result of the three-dimensional ordering of molecules in a crystal is the appearance of
plane faces. Perhaps the most obvious property of a crystal is its macroscopic geometrical
shape.
Are all solids crystalline?
23. Solids which are not crystals
Material such as glass is a crystalline solid in the same sense that calcite is. Both calcite and
glass are hard and transparent to light. But although glass may fracture, it does so in an
irregular manner.
What is glass made out of?
The structure of glass comprises long macromolecules of silicon dioxide which have cooled in
a random manner. Glasses do not have a regular, three-dimensional structure and so they can
not be referred to as crystalline.
This can be verified by the fact that glasses do not show a sharp melting point but become
progressively more fluid. Since the thermal energy available as the glass cools is not sufficient
to allow the polymer to form a regular configuration, the randomness of the liquid state is
frozen in.
24. Silicon
Oxygen
Solids in which there is no long-range order in the positions of the constituent atoms or
molecules are referred to as amorphous. Amorphous solids can be made from solids that
normally crystallize by rapidly cooling molten material.
25. Crystal defects
Long-range implies an order over about 106 spacing. In fact, it is rare to find crystals which
preserver perfect ordering over macroscopic distances such as may be measured with ease
using ordinary laboratory equipment.
Once a crystal is regular over 106 spacing, it will exhibit the properties of a crystalline solid,
but thereafter it is possible for various defects to be present as long as the perturbing effect of
these defects dos not have too large an effect.
For example, it is quite possible for an array of 106 molecules to have one vacant molecular
site without disturbing the overall structure significantly. If a solid is composed of many
aggregates of a volume such that the order is perfect over about a million spacing, then each
of these volumes is termed a crystallite. Metals are generally of this form.
What is the upper limit of long-range?
