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

TL;DR

This study optimized conditions for carboplatin binding to a model protein without NaCl, used multiple criteria to determine diffraction resolution, and observed time-dependent binding of carboplatin to specific protein sites.

Contribution

It demonstrates non-NaCl crystallization conditions for carboplatin binding and compares various criteria for resolution limit determination in X-ray crystallography.

Findings

Carboplatin binds to His-15 of HEWL under non-NaCl conditions.

Binding of carboplatin increases from one to two molecules over five weeks.

Using multiple criteria improved the resolution of diffraction data.

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 2.0{\AA}, compared to the data only being able to be processed to 3.0{\AA} 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 2.3{\AA}. Interestingly this was compared with results from the EVAL software package which gave a resolution limit of 2.2{\AA} solely using <I/sigI> crossing 2, but 2.8{\AA} based on the Rmerge values (60%). The structural results showed that carboplatin bound to only the N{\delta} 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.