The high-throughput protein-to-structure pipeline at SECSG.

Using a high degree of automation, the crystallography core at the Southeast Collaboratory for Structural Genomics (SECSG) has developed a high-throughput protein-to-structure pipeline. Various robots and automation procedures have been adopted and integrated into a pipeline that is capable of screening 40 proteins for crystallization and solving four protein structures per week. This pipeline is composed of three major units: crystallization, structure determination/validation and crystallomics. Coupled with the protein-production cores at SECSG, the protein-to-structure pipeline provides a two-tiered approach for protein production at SECSG. In tier 1, all protein samples supplied by the protein-production cores pass through the pipeline using standard crystallization screening and optimization procedures. The protein targets that failed to yield diffraction-quality crystals (resolution better than 3.0 A) become tier 2 or salvaging targets. The goal of tier 2 target salvaging, carried out by the crystallomics core, is to produce the target proteins with increased purity and homogeneity, which would render them more likely to yield well diffracting crystals. This is performed by alternative purification procedures and/or the introduction of chemical modifications to the proteins (such as tag removal, methylation, surface mutagenesis, selenomethionine labelling etc.). Details of the various procedures in the pipeline for protein crystallization, target salvaging, data collection/processing and high-throughput structure determination/validation, as well as some examples, are described.

Isolation, crystallization and preliminary X-ray analysis of a methanol-induced corrinoid protein from Moorella thermoacetica.

A corrinoid protein was induced and overexpressed in methanol-grown cells of the thermophilic anaerobic bacterium Moorella thermoacetica. The protein was purified from cytosolic extracts. After screening for crystallization conditions and optimization, crystals were obtained that diffracted strongly on a rotating-anode X-ray source. A diffraction data set was collected and processed including reflections to 1.9 A resolution. Reflections were indexed in a primitive orthorhombic cell with unit-cell parameters a = 55.69, b = 62.74, c = 34.54 A. N-terminal amino-acid sequencing indicates that the crystals contain a C-terminal fragment of the protein.