Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline

Abstract

Structural genomics is emerging as a principal approach to define protein structure-function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.

Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS

“…Proteome-scale crystallography is being pursued by several groups, including the Joint Center for Structural Genomics (JCSG) (1)(2)(3). These efforts have benefited greatly from recent technology enhancements in protein expression and crystallization.…”

Crystal structure of an Apo mRNA decapping enzyme (DcpS) from Mouse at 1.83 Å resolution

“…The protein was crystallized using the nanodroplet vapor diffusion method 13 with standard JCSG crystallization protocols. 5 The crystallization reagent contained 20% polyethylene glycol (PEG)-3350 and 0.2 M potassium fluoride at pH 7.2. 15% ethylene glycol (final concentration) was included as a cryoprotectant.…”

“…Here, we report the crystal structure of DcpS from Mouse determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG). 5 The crystal structure of Mouse DcpS [ Fig. 1(A)] was determined to 1.83-Å resolution by the molecular replacement (MR) method using a search model constructed from human DcpS [Protein Data Bank (PDB) code: 1st0], with a sequence identity of 89%.…”

Practical applications of structural genomics technologies for mutagen research

“…The first phase of funding established a number of competing vertically integrated, multidisciplinary centers that had as their main objectives accelerating the rate of structure determination and greatly reducing the cost of determining crystal structures, largely by realizing the economy of scale and industrialization [9]. Phase I PSI centers focused largely on developing structural genomics pipelines (figure 2).…”