| In my personal opinion, protein crystallography as a field has undergone some growing pains recently in trying to rediscover itself, leading to both conflict and growth. Whereas laboratories were once based around solving the structure of a single protein, changes in the field have led more towards a high throughput approach. These high throughput laboratories solve as many structures as possible from a given target set, capitalizing on their ability to pass each protein through a predetermined sets of conditions. If a structure was solved, biological relevance might be studied for a time before the protein was annotated, shelved, and work proceeded on another protein.{09}The goal was to test the capabilities of the crystallographic technologies, however not look for a specific piece of biological information. Such was the case with the Thermotoga maritima proteome with the JCSG, where I began my education in applied protein crystallography working on a structural solution of TM0293, gamma-glutamyl phosphate reductase. The lessons, methods, and much of the equipment from this project were then used on a similar collaborative project, to solve the structures of the proteins in the SARS coronavirus proteome. Several significant differences existed in the SARS project, as here the scientific scope of the work meant that proteins that did not express or crystallize under the regular battery of conditions could not be passed over; here, functional and biological information were relevant components that helped drive target identification. As it became clear that the initial high throughput approach was going to have a very low rate of success, the focus changed from screening many variants of a given protein in tandem, to focusing intensively on a few proteins at a time. Viral proteins were found to require much more specialized treatment. Using the increasing amounts of structural and functional data available from other labs in the FSPS and the literature, it was apparent that some of the SARS proteins depended on other proteins to fold stably, and multiple proteins might need to be present in order to solve a structure. Through the time spent with the FSPS, I have explored the high throughput cloning and microexpression of the SARS-CoV, worked on the peptide synthesis of targets too small to be expressed in E. coli or baculovirus, cloned and expressed SARS nsP7 and SARS3a, expressed human cyclophylin A for interaction studies with the nucleocapsid, and expressed and purified the SARS 3CL protease. |
