A system for the rapid and systematic characterization of protein crystals

Abstract

We have initiated a structural genomics project based on selected protein families, with the representatives for structural studies obtained from the genome of E. coli. A total of 160 genes have been cloned to date, with 126 successfully overexpressing soluble protein with at least one fusion system. We are currently applying robotics methods allowing cloning in an automated manner. Target genes have been cloned as N-terminal fusions with GST, (His)6, or (His)8 affinity tags. Over 60 proteins have so far been purified to homogeneity. Purified proteins are further characterized for homogeneity and suitable solution properties using a combination of dynamic light scattering and electrophoretic methods. Purified protein samples are screened for initial crystallization conditions in 96-well format using a sparse-matrix approach. A specialized database has been developed to follow the progress of various proteins and to store all relevant experimental data (http://sgen.bri.nrc.ca/bsgi). Web-based software for displaying and searching the information from this local database and for tracking deposited structures in the PDB as well as the progress of other structural genomics projects has been developed. To date, crystals have been obtained for 37 of the purified proteins. of these, diffraction quality crystals were obtained for 16 proteins. Using SeMet-substituted proteins we have determined the structures of 13 of these proteins by MAD phasing. Structures determined include 2amino-3-ketobutyrate CoA ligase, part of the threonine salvage pathway, MoeA protein, involved in molydopterin biosynthesis, histidinol phosphate aminotransferase and L-histidinol dehydrogenase, two enzymes associated with histidine biosynthesis, and RsuA, a 16S rRNA psuedouridine synthase.