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Phase problem: Difference between revisions
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"Phase problem" refers to the fact that in X-ray analysis of macromolecules, phases cannot be measured, but have to be calculated. This is a problem because both the amplitudes (which are the result of measurement) ''and'' phases are needed to compute the electron density (see K. Cowtan's [http://www.ysbl.york.ac.uk/~cowtan/fourier/fourier.html Book of Fourier]), and the procedures for calculating phases | "Phase problem" refers to the fact that in X-ray analysis of macromolecules, phases cannot be measured, but have to be calculated. This is a problem because both the amplitudes (which are the result of measurement) ''and'' phases are needed to compute the electron density (see K. Cowtan's [http://www.ysbl.york.ac.uk/~cowtan/fourier/fourier.html Book of Fourier]), and the procedures for calculating phases depend on requirements that may or may not be fulfilled in a specific case. | ||
The phase problem may also be solved with the positioning of a similar molecule in the correct orientation and location in the asymmetric unit of the (crystallized) unknown structure. This approach is | == [[Experimental phasing]] == | ||
A recent introduction into the principles of phase calculation with the help of experiments (SIR/MIR/SIRAS/MIRAS/SAD/MAD) can be e.g. found in Taylor, G. (2003) The phase problem. Acta Cryst D59, 1881-1890 [http://journals.iucr.org/d/issues/2003/11/00/ba5050/index.html]. Requirement for applicability of [[experimental phasing]] is [[isomorphism]] between heavy-atom derivative and native (i.e. the macromolecule itself, and the crystal's cell should be unchanged), specific binding of heavy atom, and enough heavy-atom incorporation into the crystal. | |||
== [[molecular replacement|Molecular Replacement]] (MR) == | |||
The phase problem may also be solved with the positioning of a similar molecule in the correct orientation and location in the asymmetric unit of the (crystallized) unknown structure. This approach requires that the known molecule is similar enough to the crystallized one. A rule of thumb is that MR will succeed if the r.m.s. deviation of backbone atoms is about 1.5 A or less. Usually this is fulfilled if the percentage of sequence identity is more than, say, 30% . | |||
== [[direct methods|Direct Methods]] == | |||
The solution of the phase problem with the help of [[direct methods|Direct Methods]] was awarded with the Nobel Prize in 1985 ([http://www.iucr.org/people/nobel-prize]). In the context of macromolecular crystallography, this approach is used in substructure determination, and at very high resolution (beyond 1.2A). | The solution of the phase problem with the help of [[direct methods|Direct Methods]] was awarded with the Nobel Prize in 1985 ([http://www.iucr.org/people/nobel-prize]). In the context of macromolecular crystallography, this approach is used in substructure determination, and at very high resolution (beyond 1.2A). | ||