Crystallography up till now has been the most powerful technique for determining the atomic structure of proteins. However, of the 31,000 proteins that have been deposited in the Protein Data Bank (PDB) only 25 X-ray structures of unrelated polytopic membrane proteins from the inner membranes of bacteria and mitochondria, as well as from eukaryotic membranes, are available (for a regularly updated list, see http://www.mpibp-frankfurt.mpg.de/michel/pub-
Springer Series in Biophysics c.R.Mateo etal. (Ed.) Protein-Lipid Interactions © Springer-Verlag Berlin Heidelberg 2006
lic/memprotstruct.html). To understand the reasons for this limited number of membrane protein structures, the fundamentals of crystal structure determination by X-ray crystallography must be mentioned.
The first prerequisite for solving the three-dimensional structure of a protein by X-ray crystallography is a well-ordered crystal that will diffract X-rays strongly. Crystallization is usually quite difficult and obtaining such crystals is the rate-limiting step to structure determination. A pure and homogeneous protein sample is crucial for successful crystallization, and recombinant DNA techniques have been a major breakthrough in this regard. Besides, the past three years have seen some of the greatest achievements in the field of protein crystallization by way of automating and miniaturizing crystallization trials. The ability to dispense trials consisting of nanolitre volumes in a high-throughput mode has reduced the time needed to set up a series of experiments from weeks to minutes, and the subsequent phase of image capture and analysis of crystallization drops is also progressing at high speed (Luft et al. 2003). However, the crystallization problem has not been solved, and it has been estimated than only 4% of the cloned proteins in major structural genomics projects produced diffracting crystals (Chayen 2004).
X-ray waves passing through a crystal generate a diffraction pattern that depends on the reciprocal lattice. However, diffraction patterns register only the amplitude of the waves; and to get the molecular structure, one also needs to know the "phase" (the relative phase of the waves corresponding to each reflection). This information is lost in the diffraction data, and has to be inferred. This is the so-called phase problem. To overcome this problem, different phasing methods are now available; in most of them the aim is to preserve isomorphism, such that the only structural change upon heavy-atom substitution is local and there are no changes in unit-cell parameters or orientation of the protein in the crystal cell. However, single- or multiwavelength anomalous diffraction (SAD/MAD) phasing methods avoid this problem by using a single crystal. These last methods are based on the fact that for certain X-ray wavelengths, the interaction between the X-rays and the electrons of an atom causes the electron to absorb the energy of the X-ray. This produces a small change in the X-ray scattering of the atom that can be used to solve the phase problem. The introduction of more accurate detectors, as well as the use of crystal cryoprotection techniques, has now made it possible to collect diffraction intensities very accurately. Precise control of the wavelength of synchrotron radiation and routine incorporation of selenomethionine into proteins has contributed to the popularity of the SAD/MAD method of phasing. Once the phase problem is overcame the three-dimensional model of the protein can be, more or less, easily obtained.
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