Ce-SAD Phasing of Glucose Isomerase and Thermolysin Using Cu Kα Radiation

Current structural genomics projects aim to solve a large number of selected protein structures as fast as possible. High degree of automation and standardization is required at every step of the whole process to speed up protein structure determination. Phase problem is a bottleneck in macromolecular structure determination and also in model building which is a time-consuming task. The simplest approach to phasing macromolecular crystal structures is the use of a SAD signal. SAD data can be collected using the in-house copper (1.54 Å) wavelength source. Data collected using copper wavelength with the incorporation of anomalously scattering heavy metal atoms may serve as a powerful tool for structural biologists to solve novel protein structures as well where synchrotron beam line is not available. A short soak of protein crystals in heavy metal solution or by incorporating heavy atoms into the protein drop while crystallizing the protein (co-crystallization) leads to incorporation of these heavy metal ions into the ordered solvent shell around the protein surface. The present work aims to determine whether cerium ion can be successfully incorporated into the protein crystal through quick-soaking method while maintaining the isomorphism. The study also aims in understanding whether this metal ion can be used for phasing purpose. The intensity data are collected and analyzed for anomalous signal, substructure solution and the binding sites.