Computational protocol: An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

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Protocol publication

[…] The structure of the apo-CpGAPDH was solved by molecular replacement, using the entire Trypanosoma cruzi glycosomal GAPDH tetramer (1K3T) as the search model []. This model gave a clear solution in the rotation and translation functions, and the electron density maps were of sufficient quality to allow replacement of non-identical residues and rebuilding of several regions where there were deletions compared to the T. cruzi structure. Residues 185–197 could not be modeled due to very weak electron density in this area. The graphics program COOT was used for model-building [].Since crystals of the holoenzyme and the ternary complex (the C153S mutant) were isomorphous with those of the apo-CpGAPDH, the latter structure was simply placed into the cell for each structure and initially refined by rigid body techniques. The electron density for the loop containing residues 185–197 was clear in both of these structures. Difference electron density maps clearly showed the positions of the NAD molecules in both structures as well as the positions of the D-G3H molecules in the ternary complex (see later).Refinement of the apo and holoenzyme structures was performed by simulated annealing using CNS [] with the stereochemical parameter files defined by Engh and Huber []. No sigma cutoff was applied to the data. Five percent of the data were randomly selected and removed prior to refinement for analysis of the free R factor. The four subunits were restrained by the non-crystallographic symmetry during most of the refinement. The restraints were gradually relaxed as refinement proceeded and dropped completely at the final stages of refinement. The progress of the refinements was guided by the decrease in both the conventional and free R factors. Individual B-factors were included in the final refinements for each structure. In case of the ternary complex, after preliminary refinement of the polypeptide chains, residues S153 and H180 were mutated to glycine, and difference electron density maps calculated at this stage allowed us to place the side chains for these residues as well as the cofactor and the substrate unambiguously. Attempts to refine other orientations of the substrate resulted in short contacts and unsuccessful refinement. Refmac5 [] was used for further refinement of the structure. Table contains a summary of the refinement parameters for all three structures. […]

Pipeline specifications

Software tools Coot, CNS, REFMAC5
Application Protein structure analysis
Organisms Cryptosporidium parvum, Geobacillus stearothermophilus
Chemicals Glyceraldehyde 3-Phosphate, NAD