High throughput structural genomics and its general applicability to structural biology

Abstract

Over the past 7 years, the JCSG (www.jcsg.org) has evaluated, developed, adapted and integrated various methodologies and technologies into a high throughput (HT) production pipeline for all steps from target selection, cloning, expression, crystallization to structure determination. The pipeline which was initially developed using the full proteome screen of T. maritima as our main source of targets, now forms the base of our current production pipeline. As one of the 4 PSI production centers, we have focused on large Pfam families without structural coverage, as well as on proposing and validating new protein families, that have recently been add to Pfam. Our biomedical theme project revolves around, The Central Machinery of Life, proteins that are conserved in all kingdoms of life. New and exciting projects in our target portfolio are focused on metagenomes, in particular, the Global Ocean Sampling (GOS) and human microbiomes. To date. we have processed over 11.000 targets, solved over 435 structures and deposited over 405 in the PDB. The T. maritima effort has resulted in 163 structures (total unique structures in PDB: 282) that now account for a 15% direct structural coverage of its predicted soluble proteome. Moreover, of its 1877 predicted ORFs, only 95 proteins (5% of the predicted soluble) are currently without fold predictions. As a result of processing such a large number and variety of targets through our HT pipeline, we have been able to test and validate various strategies to increase efficiency, improve yield, and lower cost per structure. This large scale focused studies have resulted in adding a number of routine salvage pathways to our pipeline. Many of these advances in technologies and methodologies can easily be implemented on a smaller scale in individual structural biology labs. The JCSG, located at The Scripps Research Institute, Genomic Institute of the Novartis Research Foundation, U.C. San Diego, Burnham Institute, and the Stanford Synchrotron Radiation Laboratory/Stanford University, is supported through the NIH Protein Structure Initiative (U54-GM074898), (www.nigms.nih.gov/psi).