Protein crystallography direct methods

In small-molecule crystallography the phase problem was solved by so-called direct methods (recognized by the award of a Nobel Prize in chemistry to Jerome Karle, US Naval Research Laboratory, Washington, DC, and Herbert Hauptman, the Medical Foundation, Buffalo). For larger molecules, protein aystallographers have stayed at the laboratory bench using a method pioneered by Max Perutz and John Kendrew and their co-workers to circumvent the phase problem. This method, called multiple isomorphous replacement... 

The method we will not spend much time discussing, as it is currently not of much use in protein crystallography, is the direct method. This is the method of choice for determining the structures of small molecules, but as yet is only of limited value in protein crystallography. Much work is being carried out on this particular problem and advances have been made, but are not enough to make it practical for most protein crystallography applications. 

Gilmore, C.J., Nicholson, W.V., and Dorset, D.L., (1996) Direct methods in protein electron crystallography the ab initio structure determination of two membrane protein structures in projection using maximum entropy and likelihood, Acta Cryst A52, 937-946. 

Karle J (1989) Direct methods in protein crystallography. Acta Cryst A45 765 -780... 

Hauptmann, H. The Direct Methods of X-Ray Crystallography. Sdence 23>% 178-183 (1986). [A discussion of improvements in methods of doing the calculations involved in determining protein strucmre based on a Nobel Prize address. This article should be read in connection with the one by Karle, and it provides an interesting contrast with the articles by Perutz, both of which describe early milestones in protein crystallography.]... 

For macromolecules such as proteins, the numbers of atoms that compose molecules are huge, therefore the crystal cells contain large numbers of atoms. It is not possible to apply the methods for small molecules, such as the direct method or Patterson map searching, in the structure determinations of proteins. The methods for retrieving the phases of protein crystal diffractions are molecular replacement, isomorphous replacement and anomalous scattering. In recent years, the direct method, which has been widely and successfully used in the determination of small-molecule structures, has also been applied in protein crystallography. 

Thus, although the nmr methods are important for comparison of the structure of proteins in the solid state and in solution, they are also of importance in areas where X-ray crystallography can provide little information. One of these areas concerns the time dependence of protein structure, for molecular motion over a wide range of time scales can be detected. Table IV indicates the methods and references to these studies. The range of the nmr technique is from about 10 10 s to slower than 10 s. Thus, although more restricted than X-ray crystallography in direct structure determination, the nmr studies can check the solution structure and complement the diffraction studies once the overall similarity between the solid and solution structure is proved. In this way nmr relates the static picture of a protein structure to the kinetic data of solution chemistry. 

Crystal structure of a protein molecule can also be determined by x-ray crystallography. Purified protein is crystallized either by batch methods or vapor diffusion. X-rays are directed at a crystal of protein. The rays are scattered depending on the electron densities in different positions of a protein. Images are translated onto electron density maps and then analyzed computationally to construct a model of the protein. It is especially important for structure-based drug designs. 

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