An approach to solving the phase problem in protein structure
determination by comparing structure factors collected at different
wavelengths, including the absorption edge of a heavy-atom scatterer.
Also known as multiple-wavelength anomalous diffraction or
multiwavelength anomalous dispersion.
The 'normal' atomic scattering factor f0 describes the strength of X-rays scattered from the electrons in an atom assuming that they are free oscillators. Because the scattering electrons are in fact bound in atomic orbitals, they act instead as a set of damped oscillators with resonant frequencies matched to the absorption frequencies of the electron shells. The total atomic scattering factor f is then a complex number, and is represented by the sum of the normal factor and real and imaginary 'anomalous' components:
f = f0 + f' + if''.
A consequence of the wavelength dependence of anomalous dispersion is that the structure factors will be significantly perturbed, both in amplitude and in phase, by resonant scattering off an absorption edge. Hence, if diffraction is carried out at a wavelength matching the absorption edge of a scattering atom, and again at a wavelength away from the absorption edge, comparison of the resulting diffraction patterns will allow information to be extracted about the phase differences. For suitable species, the effect is of comparing a native molecule with a strictly isomorphous derivative (and in such cases phase determination and improvement are similar to isomorphous replacement methods).
The technique, often using tunable synchrotron radiation, is particularly well suited to proteins where methionine residues can be readily replaced by selenomethionine derivatives; selenium has a sufficiently strong anomalous scattering effect that it allows phasing of a macromolecule.
The method of Multiple wavelength Anomalous Diffraction (MAD) is most applicable to problems where there are no available separate native protein diffraction data, e.g. for metallo-proteins where a heavy atom is already bound in the native structure, or to cases where derivative crystals are non-isomorphous and are therefore unsuitable for phasing via isomorphous replacement.
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The 'normal' atomic scattering factor f0 describes the strength of X-rays scattered from the electrons in an atom assuming that they are free oscillators. Because the scattering electrons are in fact bound in atomic orbitals, they act instead as a set of damped oscillators with resonant frequencies matched to the absorption frequencies of the electron shells. The total atomic scattering factor f is then a complex number, and is represented by the sum of the normal factor and real and imaginary 'anomalous' components:
f = f0 + f' + if''.
A consequence of the wavelength dependence of anomalous dispersion is that the structure factors will be significantly perturbed, both in amplitude and in phase, by resonant scattering off an absorption edge. Hence, if diffraction is carried out at a wavelength matching the absorption edge of a scattering atom, and again at a wavelength away from the absorption edge, comparison of the resulting diffraction patterns will allow information to be extracted about the phase differences. For suitable species, the effect is of comparing a native molecule with a strictly isomorphous derivative (and in such cases phase determination and improvement are similar to isomorphous replacement methods).
The technique, often using tunable synchrotron radiation, is particularly well suited to proteins where methionine residues can be readily replaced by selenomethionine derivatives; selenium has a sufficiently strong anomalous scattering effect that it allows phasing of a macromolecule.
The method of Multiple wavelength Anomalous Diffraction (MAD) is most applicable to problems where there are no available separate native protein diffraction data, e.g. for metallo-proteins where a heavy atom is already bound in the native structure, or to cases where derivative crystals are non-isomorphous and are therefore unsuitable for phasing via isomorphous replacement.
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