The phase problem arising in a structure analysis of protein crystals was initially solved by soaking the crystals in solutions of heavy atom compounds.4'8'11 This is possible because the crystals contain about 50% solvent in interconnected channels so that molecules with masses below about 1000 Da tend to reach every available protein surface patch. Heavy atoms sitting at defined places on the proteins can be located in so-called difference-Patterson maps and subsequently used for the phase determination. Useful heavy atoms should have more than about 50 electrons. Various mercury and platinum compounds are most frequently applied. Other popular compounds contain uranium, lead, thallium, iridium, osmium, tungsten, tantalum, numerous lanthanides, barium, xenon, and iodine atoms. In general, phase determination through heavy atom soaking is a most tedious task because binding of these compounds tends to modify slightly the packing scheme of the native protein and also its conformation. Consequently, the derivatized crystals tend to become nonisomorphous with respect to the native crystal, which is an outcome that destroys the phase information.
As a further general experience, most heavy atom compounds fail to bind at a defined, recurring position in the crystal and thus cannot contribute to phasing. Alternatively, many of the compounds break the crystals, presumably because they bind at packing contacts. A somewhat special heavy atom agent is xenon, which may fill holes in the nonpolar cores of proteins without too much destruction. However, its application requires a special apparatus that bathes the crystal in 10-20 bar of this noble gas.66 Holes and thiols in the core of a protein may also be reached by methylmercury compounds. In another approach, crystals were bathed in highly concentrated bromide salt solutions and shock-frozen to 100 K, which, surprisingly, left some of the bromides at defined positions on the protein surface.67 These bromides were then applied for phase determination. In summary, the defined binding of heavy atoms in crystals remains an undertaking with unpredictable outcome and usually involves many unsuccessful experiments.
Once a protein structure is established, the analysis may change to address functional questions. For this purpose, protein crystals are often soaked with known ligands of the protein under scrutiny. Such an analysis may have important commercial consequences, for instance, for the pharmaceutical industry,68 or it may help clarifying chemical reactions involving the protein.69 Once the basic structure of the crystallized protein is known, such ligand-soaking experiments are no longer disturbed by nonisomorphism because each analysis is independent of all others. However, the experiments may become tiresome because in most cases soaking breaks the crystals, in particular if ligand binding induces conformational changes that deteriorate the packing contacts.
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