The Crystallization Expert System Xtaldb, and its Application to the Structure of the 5'- nucleotidase YfbR and Other Proteins

Abstract

Growing crystals is a critical step in the process of determining the 3-D structure of macromolecules by X-ray crystallography. While significant progress has been made in analyzing quantitatively crystallization experiments, in general, crystallization of biological macromolecules remains more of an art than a science. This work presents Xtaldb, an expert system for the quantitative analysis of crystallization experiments.Xtaldb provides tools for efficiently designing crystallization screens, tracks in a semiautomatic manner most of the parameters of the experiments, and provides sophisticated search, analysis, and data graphing tools. The algorithm used to produce balanced random screens is the fastest and most robust available. Xtaldb was tested on a set of six novel proteins that had failed to produce diffraction-quality crystals in a high-throughput structure determination pipeline. Of them, five yielded crystals diffracting to 3.5 Å or better and three 3-D structures were elucidated. One of the three proteins was YfbR, a member of the HD-domain phosphohydrolase superfamily. Structural analysis of the 3-D structure and biochemical work confirmed phosphohydrolase activity. Further studies by a collaborator demonstrated that YfbR is a 5'-deoxynucleotidase. Xtaldb was used to produce two crystals of catalytically inactive YfbR mutants in the presence of metal cofactor and the substrates TMP or dAMP. The structures of the complexes explained the mechanism of the unique pattern of substrate selectivity, further supported by computational docking studies. The complex structures suggested a plausible atomic mechanism of catalysis for the enzyme, the first proposed mechanism for an HD-domain phosphohydrolase based directly from enzyme-substrate complex structures.