Isomorphous replacement practice

MIR), requires the introduction of new x-ray scatterers into the unit cell of the crystal. These additions should be heavy atoms (so that they make a significant contribution to the diffraction pattern) there should not be too many of them (so that their positions can be located) and they should not change the structure of the molecule or of the crystal cell—in other words, the crystals should be isomorphous. In practice, isomorphous replacement is usually done by diffusing different heavy-metal complexes into the channels of preformed protein crystals. With luck the protein molecules expose side chains in these solvent channels, such as SH groups, that are able to bind heavy metals. It is also possible to replace endogenous light metals in metal-loproteins with heavier ones, e.g., zinc by mercury or calcium by samarium. 

In order to resolve the phase ambiguity from the first heavy-atom derivative, the second heavy atom must bind at a different site from the first. If two heavy atoms bind at the same site, the phases of will be the same in both cases, and both phase determinations will provide the same information. This is true because the phase of an atomic structure factor depends only on the location of the atom in the unit cell, and not on its identity (Chapter 5, Section III.A). In practice, it sometimes takes three or more heavy-atom derivatives to produce enough phase estimates to make the needed initial dent in the phase problem. Obtaining phases with two or more derivatives is called the method of multiple isomorphous replacement (MIR). This is the method by which most protein structures have been determined. 

The Patterson synthesis (Patterson, 1935), or Patterson map as it is more commonly known, will be discussed in detail in the next chapter. It is important in conjunction with all of the methods above, except perhaps direct methods, but in theory it also offers a means of deducing a molecular structure directly from the intensity data alone. In practice, however, Patterson techniques can be used to solve an entire structure only if the structure contains very few atoms, three or four at most, though sometimes more, up to a dozen or so if the atoms are arranged in a unique motif such as a planar ring structure. Direct deconvolution of the Patterson map to solve even a very small macromolecule is impossible, and it provides no useful approach. Substructures within macromolecular crystals, such as heavy atom constellations (in isomorphous replacement) or constellations of anomalous scattered, however, are amenable to direct Patterson interpretation. These substructures may then be used to solve the phase problem by one of the other techniques described below. 

Methods for treating isomorphous replacement data, in practice, are mathematical in nature, employ probability and statistics to deduce the best possible phases hia for each Ff,ki, and assign to that phase some measure of its precision. In the Fourier syntheses used to produce electron density maps, the individual terms are then weighted with their likelihood of being accurate or according to their precision. 

Usually, isomorphous replacement methods are coupled with a density modification process called solvent flattening. The solvent flattening process was made practical by the introduction of the ISIR/ISAS program suite (Wang, 1985) and other phasing programs such as DM (CCP4,1994) and PHASES (Furey and Swaminathan,... 

In practice the mass ratio of magnesium to calcium used is between 2 1 and 4 1, and depends on the relative cost of the reagent metals. The process is promoted by the addition of antimony, which can also form intermetallic compounds as a replacement for bismuth, and is necessary to achieve low levels of residual bismuth. It is suggested that the antimony compound is isomorphous with the bismuth compound and a solution of the two causes a lowering of the activity of the bismuth complex (Moodie, 1976). 

The crystallinity measurements are of little significance in this case due to the practically perfect isomorphism 114,167). Both homopolymers have 7/2 helices, and hexene-1 readily replaces 4-methylpentene-l units in the lattice 114), This fact expldns linear dependence of the composition on crystallinity (Fig. 11). 

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