IMPROVING BROMIDE SOAKS FOR SOLVING THE PHASE PROBLEM IN CRYSTALLOGRAPHY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There are number of mammalian proteins that cannot be produced with Se-Methionine bacterial expression systems. For those proteins the phase problem could be solved by adding heavy atoms and use their differential scattering to obtain phase information and generate electron density. The method of halide soaking pioneered by Z. Dauter (NSLS) is one alternative to the Se-Met phasing. Short soaking of the protein crystals with high concentration bromine or iodine solutions results in a surface bound ions. Another universal phasing method is to introduce inert gases such as krypton and xenon by pressurizing protein crystals (developed at SSRL). If the proteins have some hydrophobic cavities the inert gases typically bind in them. Unfortunately, these modification methods suffer from low efficiency. Halide soaking often stresses the protein crystal lattice due to differences in osmotic pressure. Inert gas pressurized crystals often loose diffraction and have higher mosaicity than the initial crystals, mostly likely due to changes in hydration. An improved soaking scheme guided toward decreasing the deteriorating effects of osmosis or dehydration was applied to lysozyme (high resolution; 1.7 A), beta lactamase (medium resolution; 2.3 A), and thymidylate synthase complementary protein TM0449 (low resolution; 3.8 A). All crystals were treated with a rubidium bromide (RbBr) and glycerol containing solution resulting in ice-free derivatized frozen crystals. One lysozyme crystal was pressurized with Kr in addition to the RbBr treatment. These elements are accessible for MAD data collection at SSRL beam lines 9-1, 9-2, and 11-1. Preliminary analysis of the data indicates there is: (a) preserved resolution and mosaicity during the soak procedure; (b) significant binding of Rb and Br ions for all proteins; (c) automatic full model building for the high resolution data (lysozyme), and partial model building for the medium resolution data (beta lactamase).