Radiation Damage on Thaumatin: A Case Study of Crystals That Are Larger Than the Microfocusing X-ray Beam

Abstract

Microfocusing X-rays direct high-density photons on crystal samples and can enhance the diffraction limit and quality of collected data. However, these intense X-rays can cause radiation damage to the sample, which often results in undesirable structural information. Accordingly, a data collection strategy that minimizes radiation damage is critical to obtaining accurate structural information. In this study, radiation damage in single-point data collection was investigated at two different X-ray exposure times (1 s and 100 ms) using microfocusing X-rays and a thaumatin crystal larger than the beam. The data collection statistics showed that the diffraction intensity of the Bragg peak did not gradually decrease until the crystal rotation reached 180°, and it significantly decreased after exceeding this value. Thaumatin structures exposed to X-rays for 1 s (Thaumatin1s) and 100 ms (Thaumatin100ms) were determined at 1.13 Å resolution. The temperature factors for Asp60, Arg119, Lys163, and Lys187 of thaumatin were increased by radiation damage. Specific radiation damage was observed at the disulfide bond in Thaumatin1s but was negligible in Thaumatin100ms. Splitting and reprocessing Thaumatin100ms showed that electron density maps with minimal radiation damage can be obtained when using minimal data that satisfy the completeness, I/sigma, and CC1/2 parameters. These results expand our understanding of radiation damage phenomena in macromolecules and can be used for data collection applications.

Keywords

• radiation damage
• protein crystal
• X-ray
• disulfide bond
• electron density map
• thaumatin