## 4 Discussion  
In MX, various data collection strategies, such as single-point, multipoint, and helical data collection, have been reported [33]. Theoretically, to minimize radiation damage, multipoint data collection or helical data collection with minimal X-ray exposure to the same crystal volume is preferred. However, these functions may not be supported by the beamline, or single-point data collection must be performed depending on the characteristics or shape of the crystal. In such cases, it is important to conduct various experimental investigations on how to minimize radiation damage in single-point data collection. Here, we investigated radiation damage during single-point data collection when the protein crystal sample used was larger than the microfocusing beam. Generally, the radiation damage on the crystal increases as the exposure time increases, and the crystal diffraction intensity gradually decreases [33]. However, our experimental results show that if the crystal sample is larger than the X-ray, then the diffraction intensity may not necessarily show a tendency to decrease because the fresh crystal volume is continuously exposed to X-rays while the crystal is rotating during data collection. In addition, the diffraction intensity can increase more than before in the case of a region that is enlarged in a fresh crystal volume (e.g., 160\({}^{\circ}\) in Thaumatin\({}^{18}\) or 30\({}^{\circ}\) in Thaumatin\({}^{100 ms}\)) exposed to X-rays. On the other hand, when using a microfocusing beam, the fresh crystal volume exposed to X-rays is greater when a larger crystal is used. Thus, the volume ratio of the radiation-damaged crystal to the total diffraction information may be reduced. Therefore, large crystals must be used to achieve the effect of minimizing radiation damage, even with microfocused beams. Meanwhile, because X-rays are continuously exposed to the center of the crystal, which is aligned with the X-ray, the diffraction intensity from the crystal center theoretically decreases and radiation damage is accumulated. Accordingly, the radiation-damaged diffraction patterns from the center of the crystal and diffraction patterns from the fresh crystal volume are mixed until the crystal rotation reaches 180\({}^{\circ}\). However, because there is no fresh crystal volume after 180\({}^{\circ}\), significant radiation damage information is included. In the total data collection statistics of Thuamatin18 and Thuamatin100 ms, no numerical difference that can be regarded as radiation damage was found, and clear differences were observed when individual images of each data were analyzed. However, because the crystal is larger than the X-ray beam, characteristics that can be regarded as radiation damage were not found until the crystal was rotated by 180\({}^{\circ}\), and radiation damage could be distinguished after 180\({}^{\circ}\) when X-rays were exposed at least twice. For Thuamatin18, which was exposed to X-rays for a long time, a significant decrease in the intensity and number of Bragg peaks was observed after the crystal was rotated 180\({}^{\circ}\) and the Rmerge value increased, thus confirming that global radiation damage occurred. For Thuamatin100 ms, there was no noticeable deterioration in data quality as the X-ray exposure time increased in terms of the Bragg peak intensity, I/sigma, or Rmerge. Accordingly, in terms of data collection, if a large crystal with high symmetry (for example, a tetragonal space group) is exposed to a microfocusing beam, completed data collection before 180\({}^{\circ}\) rotation is possible. However, for a large crystal with low symmetry (for example, a triclinic space group), data collection over more than 180\({}^{\circ}\) of rotation is required. In this case, to reduce radiation damage, a strategy to minimize the amount of X-rays exposed to the crystal by reducing the X-ray exposure time or using an attenuator is required.