Using the Weak Scatterer Substructures
Zbigniew Dauter
Synchrotron Radiation Research Section, National Cancer Institute, Frederick and Brookhaven National Laboratory, Bldg. 725A-X9, Upton, NY 11973, USA
Recent advancements in macromolecular crystallography have made it possible to solve crystal structures of proteins from various types of the phasing signal provided by some relatively weak scatterers. The phasing signal can originate from certain scatterers naturally present in the investigated macromolecule or can be deliberately incorporated into the native crystal.
The halide cryosoaking approach is a simple method of derivatization, where the native crystals are immersed for a few seconds in solutions containing the appropriate cryoprotectant as well as the sub-molar concentration of the bromide or iodide salts. The halide ions quickly diffuse into protein crystals and take up ordered sites around the protein surface. These sites are usually partially occupied and shared with the solvent water molecules. Halide ions form hydrogen bonds as well as van der Waals interactions with the neighboring protein residues and water molecules.
Heavier halides display significant anomalous scattering properties. Bromine has the X-ray absorption edge at 0.92 Å and can be used for MAD phasing. Iodine does not have easily accessible absorption edges, but at longer wavelengths its anomalous scattering contribution is substantial and can be used for SAD or MIR phasing. In contrast to the popular SeMet derivatives, the number of halide sites cannot be predicted in advance, and usually the weak sites can be accepted for phasing as long as they increase the overall phasing power of the derivative.
Native proteins contain about 3 –4 % of cysteines and methionines, so that the anomalous signal originating from sulfurs is about 1 % of the total scattering signal of protein crystals. Phosphorus present in all nucleic acids provides the anomalous signal at about 2 % level. These signals are small, but can be successfully used for phasing if the diffraction data accuracy is sufficiently high. Several novel and test protein structures have been solved by SAD method based on the anomalous signal of sulfur as small as 0.7 % of the total diffraction.
Protein crystals irradiated by very intense X-ray beams, undergo some specific structural changes, so that the radiation damaged crystals can be treated as isomorphous derivatives. Apart from many small changes, the typically most pronounced features involve disruption of the disulfide bridges or the covalently bound halides in brominated nucleic acid bases or in iodinated tyrosines. The isomorphous signal provided by the 10 % change of the occupancy of a sulfur atom amounts to about 1.6 electron units, and in theory is stronger than the anomalous scattering contribution of the sulfur atom. It is also possible to use the combination of both, radiation damage and anomalous scattering effects for phasing crystal structures.
It may be expected that the interest in phasing methods based on weak signals originating from various weak scatterer substructures will increase, due to their experimental simplicity, especially with the availability of more and more powerful programs used for the location of substructures, the evaluation of phases and the automatic model building.
