SLIDE 13 I = | F 2 |
F(hkl) = F(hkl)⋅ei
hkl
ϕ Houston, Houston! we have a Phase Problem !
We can measure intensities, but ....
From: G. Rhodes; Crystallography Made Crystal Clear
Phasing Methods
Heavy atom method (MIR, multiple isomorphous replacement)
Each atom in the unit cell contributes to all observed reflection intensities. The basic principle of the MIR method is to collect diffraction data of several (multiple) crystals of the same protein, that share the same crystal properties (isomorphous), but differ in a small number of heavy atoms. The experimental approach is normally to soak protein crystals with diluted solutions of heavy metal compounds (e.g. mercury or platinum derivatives), that often bind specifically to certain protein residues. These additional atoms cause a slight perturbation of the diffraction intensities. To achieve a perturbation large enough to measured correctly, the added atoms must diffract strongly, i.e. elements with a high number of electrons (heavy atoms) are used. The differences of the reflection intensities can be used to locate the positions of the heavy atoms within the unit cell, which allows to estimate initial phases.
Anomalous scattering (MAD, multiple wavelength anomalous diffraction)
The MAD method is based on the capacity of heavy atoms to absorb X-rays of a specific wavelength. Near its characteristic absorption wavelength, the diffraction intensities of the symmetry related reflections (Friedel pairs, h,k,l and -h,-k,-l) are no longer equal. This effect is called anomalous scattering. The characteristic absorption wavelengths of typical protein atoms (N,C,O) are not in the range of the X-rays used in protein crystallography and therefore are not contributing to anomalous scattering. However, the use of synchrotron X-ray sources with adjustable wavelengths allows to collect diffraction data under conditions where heavy atoms exhibit strong anomalous scattering. In practice, several diffraction data sets are collected from the same protein crystal at different wavelengths. From the small differences between the Friedel pairs, the location of the heavy atoms can be determined and initial phases of the native data are estimated.
Molecular replacement (MR)
In some cases is structure to be examined is known to be very similar to an other structure, that has already been solved experimentally. This could be e.g. the same protein from an other organism or a mutant of this
- protein. In these cases the phases computed from of the known protein structure (phasing model) can be used
as initial estimates of the phases of the unknown protein.
