Chemical clustering and visualization applied to macromolecular crystallography

Many bioscience fields employ high-throughput methods to screen multiple biochemical conditions. The analysis of these becomes tedious without a degree of automation. Crystallization, a rate limiting step in biological X-ray crystallography, is one of these fields. Screening of multiple potential crystallization conditions (cocktails) is the most effective method of probing a proteins phase diagram and guiding crystallization but the interpretation of results can be consuming. To aid this empirical approach a cocktail distance coefficient was developed to quantitatively compare macromolecule crystallization conditions and outcome. These coefficients were evaluated against an existing similarity metric developed for crystallization, the C6 metric, using both virtual crystallization screens and by comparison of two related 1,536-cocktail high-throughput crystallization screens. Hierarchical clustering was employed to visualize one of these screens and the crystallization results from an exopolyphosphatase-related protein from Bacteroides fragilis, (BfR192) overlaid on this clustering. This demonstrated a strong correlation between certain chemically related clusters and crystal lead conditions. While this analysis was not used to guide the initial crystallization optimization, it led to the re-evaluation of unexplained peaks in the electron density map of the protein and the insertion and correct placement of a sodium, potassium and phosphate atoms in the structure. With these in place, the resulting structure of the putative active site demonstrated features consistent with active sites of other phosphatases which are involved in binding the phosphoryl moieties of nucleotide triphosphates. The new distance coefficient appears to be robust in this application and coupled with hierarchical clustering and the overlay of crystallization outcome reveals information of biological relevance. While tested with a single example the potential applications appear promising.

A novel architecture of 600 kDa bacterial citrate lyase complex

Citrate lyase activity exists in eukaryotes, bacteria, and archaea and plays a central role in cellular energy metabolism. There are two types of citrate lyases, ATP-dependent and ATP-independent. The latter, which exists only in bacteria, converts citrate to acetate and oxaloacetate using a two-step reaction catalyzed by a stable heterotrimeric protein complex comprising alpha-beta-gamma subunits. We selected three bacterial genomes in which the alpha-beta-gamma subunits of citrate lyase are all encoded on the chromosome in a continuous, co-cistronic geometry. The genes encoding the entire complex were subsequently amplified together using a single PCR reaction on genomic DNA. This approach enabled the entire citrate lyase complex from three organisms to be purified to homogeneity using a single step of Ni-NTA chromatography followed by gel-filtration chromatography. One of the three citrate lyase complexes purified yielded crystals diffracting to 4 Å. Six-fold non-crystallographic symmetry averaging enabled a high quality structure to be determined for the 18-subunit alpha-beta-gamma citrate lyase complex. This structure provides insight into the multistep catalytic mechanism employed by this enzyme.