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Physical Biochemistry X-Ray Crystallography 4

It is the electron density that gives rise to the 3D structure

The intensity of the diffraction spots are known, however we do not know the phase of the wave,
which is required to calculate the electron density in the unit cell (phase problem).

ρ(xyz) = Σh Σk Σl F(hkl) exp[-2i(hx + ky + lz)]


REQUIRED?

SLIDE 16

ρ = electron density
x, y, z = point where density has to be calculated
|F| = magnitude of structure factor
|F| = square root of intensity
h, k, l = indices of diffraction spots

Obtaining Phase Information:


For small molecules this can be done directly using a Patterson map or ‘direct methods’
This cannot be done for proteins

Isomorphous replacement method


Depends on two crystals having the same structure, but differing by a few heavy metal atoms
Crystals are soaked in heavy atom solutions such as HgCl 2, the heavy atoms will bind to the same
specific places on each protein. Hg interacts well wit cysteine.
Although there are only a few heavy atoms (HA), each has a large number of electrons and they
contribute appreciably to the scattering.
The diffraction intensities from the derivatised crystal will differ slightly to those from the native
crystal

It is important to know the positions of the heavy atoms and the differences in intensities between
the diffraction patterns of the two structures.
It is then possible to determine approximates phases (), which can be used to calculate an initial
electron density (ρ).

Not used so often anymore.

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Physical Biochemistry X-Ray Crystallography 4

Multi-wavelength Anomalous Diffraction (MAD)


Similar to MIR
MAD relies on the fact that some Has have an absorption edge (wavelength needed to knock an
electron out of the atom) close to the wavelengths used in X-ray crystallography (0.7-2.0 Å).
Close to the absorption edge, small changes in  give rise to significant changes in the phase of
the scattered wave. This is known as anomalous dispersion.

The method requires access to a synchrotron with a tuneable radiation source, so the wavelength
of X-rays used can be changed.

Friedel’s Law is only approximately correct, and the relationship it describes can be changed
depending on the wavelength used for detection. Differences can be generated by using HAs,
which can be maximised using different wavelengths.
HA such as selenium cause anomalous scattering of X-rays

Selenomethionine Substitution
Methionine synthesis auxotroph is used to generate a protein where the sulphur of Met is replaced
by the HA selenium. The anomalous difference effect in MAD is then used for phase
determination.

Molecular Replacement
If a closely related structure is known, or a previous model in a different crystal form is available,
the molecule can be located in the unit cell and phases determined from the model.

A similar protein of known structure is used to find structural homology with the protein of interest
You can then ‘search’ within the crystal lattice to find a position that would allow it to be located.
Electron density maps can be calculated for a known model.

One the position is know the phases can be calculated, along with the electron density maps.

Building the Structure:


Once phase information has been determined, an electron density map can be calculated
Atoms are placed in the centres of peaks, leading to a model of the structure
The model is improved by computer software (before computers were done in 3D by hand)
Several cycles of model building and refinement are required to obtain the best possible model

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Physical Biochemistry X-Ray Crystallography 4

Resolution
Very important in crystal structure determination

 High (better than 2.0 Å)


o Easy to interpret 4Å
Map
o Almost ‘atomic’
o Holes in aromatic groups
o Ordered water molecules
 Medium (2.5 - 3.5 Å)
o Sidechains become visible
 Low (below 4.5 Å)
o Overall shape of molecule


Map are calculated using the final, refined phases Map
Initial ,maps have large phase errors and are far worse

Refinement
Model improvement - manual model building is not sufficient to build a completely accurate model
Refinement aims to adjust the atomic model so that it gives a better fir to the observed data

Minimizing the differences between observed intensities, and the intensities calculated from the
model, my modifying the model.
Stereochemical restraints are applied – model must have regular bond lengths and angles like all
other proteins do.
Computational process that is more objective than model building
Successful structure determination requires several inter-leaved rounds of model building and
refinement.
R factor is a measure of the quality of the structure

Observations / parameters ratio is important, and is often a problem in protein crystallography as


there are so many parameter that have to be refined at one time
This problem is solved using stereochemical information

The positions of atoms need to be well defined in 3D as well as the B-factor (a measure of how
dynamic / flexible a part of the structure is).

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Physical Biochemistry X-Ray Crystallography 4

Stereochemical Restraints
Used to increase the observations / parameters ratio
A lot is known about protein structure from small molecules crystallography and from the
thousands of protein structures in the Protein Data Bank (PDB)
 Bond lengths
 Bond angles
 Dihedral angles
 Chiral centres
 Planar groups
 Van der Waals Radii

Using the database of structures we can get an idea of what makes up ideal geometry. During
refinement the structure can be restrained to conform to this ideal.

Once the initial model has been built, computer programs move the atoms into better positions in
the electron density, and ensure that all bond lengths and angles are within the expected values.

The aim is to minimise the difference between the observed amplitudes seen in the diffraction
pattern (|Fobs|) and the diffraction pattern that would be expected from the model (|
Fcal|)

ΔF = |Fobs| - |Fcal|

Fcal = Σ fje2i(hxj + kyj + lzj)

f = scattering factor associated with each


type of atom
j = index over all atoms

Refinement Statistics
The most commonly used measure of the progress of refinement is the R value

A good R value for a protein is < 0.2 (20%) but its better if the value is smaller

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Physical Biochemistry X-Ray Crystallography 4

Free R reduces bias


5% of data is not used in the refinement process, it is used to calculate the Free R value only, an
independent way of checking is a model is correct or not.

Ramachandran Plot
Because of steric clashes, only certain combinations of torsion angles are allowed
The plot can be used to plot the allowed combinations in the y,f plane

Planar peptide bond has ψ and Φ angles

Allowed regions are in yellow


Favoured regions are in red

Certain structural motifs will predominate


in different regions of the Ramachandran
plot.

The model can be run through a


Ramachandran plot program to see if
there are any disallowed regions.

We expect to find 90+% of residues to


fall into the red regions.

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