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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).
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
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).
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Physical Biochemistry X-Ray Crystallography 4
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.
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Physical Biochemistry X-Ray Crystallography 4
Resolution
Very important in crystal structure determination
2Å
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
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|
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
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
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