Carboplatin binding to a model protein in non-NaCl conditions to eliminate partial conversion to cisplatin, and the use of different criteria to choose the resolution limit

Abstract

Hen egg white lysozyme (HEWL) co-crystallisation conditions of carboplatin without sodium chloride (NaCl) have been utilised to eliminate partial conversion of carboplatin to cisplatin observed previously. Tetragonal HEWL crystals were successfully obtained in 65% MPD with 0.1M citric acid buffer at pH 4.0 including DMSO. The X-ray diffraction data resolution to be used for the model refinement was reviewed using several topical criteria together. The CC1/2 criterion implemented in XDS led to data being significant to 2.0Å, compared to the data only being able to be processed to 3.0Å using the Bruker software package (SAINT). Then using paired protein model refinements and DPI values based on the FreeR value, the resolution limit was fine tuned to be 2.3Å. Interestingly this was compared with results from the EVAL software package which gave a resolution limit of 2.2Å solely using crossing 2, but 2.8Å based on the Rmerge values (60%). The structural results showed that carboplatin bound to only the Nδ binding site of His-15 one week after crystal growth, whereas five weeks after crystal growth, two molecules of carboplatin are bound to the His-15 residue. In summary several new results have emerged: - firstly non-NaCl conditions showed a carboplatin molecule bound to His-15 of HEWL; secondly binding of one molecule of carboplatin was seen after one week of crystal growth and two molecules were bound after five weeks of crystal growth; and thirdly the use of several criteria to determine the diffraction resolution limit led to the successful use of data to higher resolution.