Effects of Radiation Damage on Metal-Binding Sites in Thermolysin

Radiation damage is an inherent problem in macromolecular crystallography because it impairs the diffraction quality of crystals and produces inaccurate structural information. Understanding radiation damage in protein structures is crucial for accurate structural interpretation and effective data collection. This study undertook X-ray data collection and structure determination of thermolysin (TLN), which contains Zn and Ca ions, by using three different X-ray doses to improve our understanding of the radiation damage phenomena on metal ions in proteins. Data processing revealed typical global radiation damage in TLN, such as an increase in unit cell volume, Rmerge value, and Wilson B-factor. An analysis of the B-factor indicated that radiation damage at the Zn and Ca sites in TLN increased with higher X-ray doses. However, the distance between the metal ions and their interacting residues in TLN was not significantly affected, suggesting that radiation damage to the metal ions has a minimal effect on these interactions. Moreover, the increase in the B-factor of the metal ions according to the X-ray dose was similar to that in the B-factor of the residues interacting with the metal ions. These results expand our understanding of radiation damage phenomena in macromolecules and can be used to improve data collection strategies.