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abstracts
Radiation damage is a limiting factor in macromolecular X-ray crystallography diffraction experiments. Global damage leads to a unit cell size increase and non-isomorphism, and to a loss of long range crystal order which is visible in the decay of the diffraction pattern and loss of high resolution information during data collection. Specific damage causes detectable changes at particularly susceptible sites in the protein structure [1,2], such as the reduction of metallo-centres, elongation and subsequent breaking of disulphide bonds and decarboxylation of aspartate and glutamate residues. Between and within these groups the decay does not happen uniformly at equal rates throughout the protein, leading to preferential specific damage. Specific damage can result in misleading biological conclusions on protein mechanism and function being drawn. We have defined a new atom-specific metric, BDamage, which facilitates the identification of protein regions susceptible to specific radiation damage as well as the quantification of the susceptibility, allowing further investigations into preferential specific damage. BDamage has been validated using a paired set of low-dose/high-dose protein structures [3]. Results show that BDamage successfully separates susceptible residues from stable parts of the protein. A non-redundant subset of previously refined structures submitted to the PDB was then analyzed for indications of specific radiation damage. BDamage indicates that the distribution of specific damage is independent of secondary protein structure or disulphide bond configuration, but shows a correlation with solvent accessibility. Results indicate a possible use of BDamage as a quality control metric for structure submission. Further research into an alternative quantification of real-space specific radiation damage, using the decay of electron density over multiple datasets, is outlined.
abstracts
Protein structures have significantly impacted and aided drug discovery efforts. However, it is not enough to know the structure of a protein; it must be the right structure. Small alteration in sequence can lead to different conformations and oligomerization states, cause changes which lead to different active site architecture and also which modify function. Protein crystallization is an essential prerequisite for the determination of protein structures by X-ray crystallography. We have obtained encouraging initial results for a hitherto unexplored crystallization method with the enzyme arylamine N-acetyltransferase from M. tuberculosis (TBNAT). Despite prolonged and varied trials to crystallize TBNAT, an important anti-tubercular drug target, no crystals were obtained. In an alternative approach, cross-seeding of TBNAT protein with micro-crystalline seeds from a homologous NAT from M. marinum (74 % sequence identity (SID)) surprisingly resulted in a single 20 micron sized TBNAT crystal that diffracted to 2.1 Å and allowed for TBNAT structure determination (Abuhammad et al., 2013). To our knowledge, cross-seeding crystallisation using homologous proteins has only been previously successful in cases with more than 85% SID. In this study, we have explored the effect of low sequence homology on cross seeding using β-lactamases with SID as low as 30%. Despite the low SIDs, the results show cross seeding leads to an increase in hits obtained, the identification of new crystallization conditions, shortening of crystallization time and an improvement in the quality of the crystals obtained.
