Structural genomics, which synergistically involves both NMR and X-ray crystallography, is a global effort aimed at the determination of three-dimensional protein structures on a genomic scale in a high-throughput mode (Berman et al., 2000
). Of particular interest in this early phase of the program is the determination of structures of proteins believed to represent new or barely known families. Every new structure deposited in the PDB increases the accuracy of homology modeling. This in turn allows better understanding of the structure–function relationships in those proteins for which structures cannot be directly determined experimentally (Stevens, 2000
). However, the effectiveness of high-throughput (HT) crystallographic analysis is impeded by several bottlenecks, including the relatively low success rate of crystallization. It is estimated that of all proteins expressed in a soluble state, only between 10 and 30% form X-ray quality crystals (Claverie et al., 2002
; Sulzenbacher et al., 2002
). The most recent statistics available from the Midwest Structural Genomics Center (http://www.mcsg.anl.gov
) show that of all successfully purified soluble proteins, less than 40% produced crystals, of which less than 60% were X-ray grade crystals that yielded useful diffraction. Thus, the first sweep of the structural genomics program is likely to harvest only the ‘low-hanging fruit’, i.e.
readily expressed, stable and easily crystallizable proteins. Unfortunately, this may leave many biologically important proteins ‘hanging high and dry’.
To circumvent one of the bottlenecks, i.e.
the crystallization problem, we proposed a novel approach to crystallization of proteins based on rational surface mutagenesis to create patches with low overall conformational entropy in order to facilitate the formation of crystal contacts (Derewenda, 2004
). This approach proved successful in a model system of human RhoGDI and allowed the crystallization and crystal structure determination of new proteins (Longenecker, Garrard et al., 2001
; Longenecker, Lewis et al., 2001
; Mateja et al., 2002
). We and others have also shown that the method is useful to generate crystal forms that diffract to much higher resolution than the wild-type protein, which may prove to be of importance in drug design (Mateja et al., 2002
; Munshi et al., 2003
We are now applying this strategy to those selected targets of the Bacillus subtilis structural genomics effort which failed to crystallize in the HT pipeline. This paper describes the crystallization by surface modification and structure determination of the product of the YdeN gene from B. subtilis (Midwest Centre for Structural Genomics code APC 1086). The protein was originally thought to have a unique sequence and potentially a novel fold. Subsequent tertiary-structure prediction revealed that it shows similarities to α/β hydrolases and this information was used to design three double mutants, including the K88A/Q89A mutant, in which residues with high conformational entropy located on putative surface loops were targeted. The K88A/Q89A double mutant produced crystals of high quality that diffracted to 1.8 Å. The structure revealed that the protein is a member of the ubiquitous α/β hydrolase family and structural considerations suggest that it is a hydrolase active on a soluble ester, possibly a thioester, but is not an interfacially activated lipase. The molecular model, including H atoms and anisotropic displacement parameters, was refined to a conventional R factor of 12.4%.