## 3 Results  
### Data Collection  
In MX with single-point collection, after aligning the crystal to the X-rays, the crystal rotates and collects diffraction data for three-dimensional structural information. When the X-rays are larger than the crystal, all crystal volumes accumulate radiation damage over the X-ray exposure time (Figure 1A). When the crystal is larger than the X-ray, the first time that the crystal is exposed to the X-ray, radiation damage occurs in the area corresponding to the X-ray path (Figure 1B). Then, when the crystal is rotated by the goniometer, the center of the crystal aligned with the X-ray beam is continuously exposed to the X-ray, whereas the crystal volume not previously exposed to the X-ray path is newly exposed to the X-ray for first time (Figure 1B). Accordingly, the X-ray dose is accumulated in the center of the crystal where the X-rays are aligned, and this area contains structural information that corresponds to radiation damage. In contrast, the fresh crystal volume that was not previously exposed to X-rays provides structural information that corresponds to minimal radiation damage. Diffraction data collected when the crystal rotates by 180\({}^{\circ}\) include structural information that corresponds to a mixture of radiation damage at the center of the crystal and diffraction information with minimal radiation damage in the fresh crystal volume. In contrast, if the rotation of the crystal sample exceeds 180\({}^{\circ}\), then the crystal is re-exposed at positions where all volumes had been previously exposed (Figure 1B). Figure 1: Schematic view of microfocused X-ray beam path on crystals that are (**A**) smaller and (**B**) larger than the X-ray beam. To understand the phenomena of radiation damage when a crystal is larger than the X-ray beam, this study exposed a thuumatin crystal (300 \(\times\) 200 \(\times\) 150 \(\upmu\)m) to a microfocused X-ray beam (3.5 \(\times\) 8.5 \(\upmu\)m, FWHM) and investigated the subsequent radiation damage. One image contains diffraction information for 1\({}^{\circ}\), and the X-ray exposure time was for 1 s (Thaumatin18) or 100 ms (Thaumatin100 ms) per image. For Thuumatin18, Bragg peaks were clearly observed up to -1.4 A in the first image. However, a decrease in the Bragg peak was observed as the X-ray exposure time increased (Figure 2A). When the oscillation reached 360\({}^{\circ}\), Bragg peaks beyond 2.1 A resolution were not clearly observed (Figure 2A). For Thuumatin100 ms, Bragg peaks were observed up to -1.5 A in the first diffraction image and continued steadily up to 1.6 A until the X-ray rotated by 360\({}^{\circ}\) (Figure 2B). This indicated that the radiation produced damage that weakened the diffraction intensity of Thuumatin18 but did not have a noticeable effect on Thuumatin100 ms.  
Next, to avoid bias in data processing, diffraction data were automatically processed using Xia2, and the standard for resolution cutoff was based on the default value of the program. Thuumatin18 and Thuumatin100 ms showed identical tetragonal space groups, and the unit cell dimensions were almost identical, with a difference of less than 0.1%. Although there was a difference in the statistical values of the two data in terms of resolution range, completeness, multiplicity, I/sigma, and CC1/2, whether one set of data was more dominant could not be determined (Table 1). Because the data statistics integrated the Bragg peak in all images (Table 1), information on the degree of radiation damage of Thuumatin18 and Thuumatin100 ms could not be obtained. Accordingly, to compare the quality of Bragg peaks according to the radiation exposure time, the number of Bragg peaks at I/sigma values of 5, 10, 20, and 30 from each of the collected images were investigated. In normal single-point mode, when exposed to X-rays, the crystal diffraction intensity gradually decreased with increasing exposure time [33].