Methods of determining three-dimensional structures of protein

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.

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).

What is the common factor in all these methods?

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

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.

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.

E2

E2-E1

E1

E2-E1

Energy

Intensity

Frequency

ΔE

ΔE

Theoretical absorption line of zero width and a line of finite width (ΔE)

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

X-ray crystallography

X-rays, discovered by Wilhlem C Rontgen, were shown to be diffracted by crystals in 1912 by

Max von Laue.

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

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?

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.

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).

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?

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.

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?

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.

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.

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?

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.

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?

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.

SiliconOxygen

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.

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?