The X-RAY CRYSTALLOGRAPHY Team was headed by George Phillips, Jr., PhD, and was one of two groups responsible for determining three-dimensional protein structures. This team focused on protein crystal production and analysis and solved over 70 X-ray structures.
The crystallomics pipeline capabilities included micro-scale screening on fluidic chips (Fluidigm Topaz), Tecan Genesis hanging / sitting drop robotics, and Bruker Crystal Farm plate handing and imaging. Over a million crystallization experiments have been conducted, with all images and their scores stored in a database for archival and research purposes. Structures are typically solved by selenomethionyl phasing using data collected at the Advanced Photon Source at Argonne National Laboratory.
During the first few years, CESG focused on Arabidopsis thaliana as the source organism for selection of fold-space targets. This choice was made due to the relatively high quality of the sequencing of that organism and the completeness of a first draft of its resulting gene model. Genes were cloned from genomic DNA in intronless or cDNA libraries. Since that time, a large number of sequence-verified cDNA’s have become available from the Mammalian Gene Collection (MGC), including primarily human and mouse full length cDNA’s, but also from the genus Ratus (rat), Bos (cow), Xenopus (frog) and Danio (zebrafish). Over 50,000 full length clones are now available. Because these genes are verified as being expressed, they represent a tremendous source of starting materials, especially for structural genomics efforts where the attrition rates in the early stages of the pipeline are high.
The Arabidopsis Biological Resource Center (ABRC) has also begun distributing full length cDNA clones, and CESG has been able to continue projects that failed at the cloning step using libraries. More recently we have extended the general strategy of using thermophilic organisms as a source of protein sequences for structure determination. We have chosen Cyanidioschyzon merolae and Galderia sulfuraria, two “red” algal species with genomic data and gene models that are under development. These organisms grow well at temperatures up to 55°C. One-hundred and ninety-two clones have been chosen based on unique structures; many have been expressed and are under evaluation. The first protein from Galderia has recently been solved. The success rate for crystallization of proteins is somewhat better than workgroups from other organisms.
CESG develops crystals of proteins using an integrated platform of micro- and macro-scaled crystallization experiments. Microfluidic chips are employed for initial screening when precious reagents are used or when the initial amount of protein is small. Sitting drop experiments are used when at least 5 mg of protein is available. The largest labor saving implementation at CESG is not automating the setup of crystallization trials, but rather is imaging the experiments: several million images have been taken since the inception of CESG. Automatic plate handling and image capture improves the quality and reproducibility of the images, which are scored by trained students on a numerical scale. Optimization of crystallization experiments follow, also using automatic image capture. CESG employs two Crystal Farm systems, one operated at 4°C and the other at 20°C.