*2.4. Data Collection*

The experiment was carried out at the SSRF beamline BL18U1. The energy used for data collection was 12.662 keV, the photon flux at the sample point was 6 <sup>×</sup> 10<sup>11</sup> phs/s, and the size of focused beam used for experiment was 20 <sup>×</sup> <sup>20</sup> <sup>μ</sup>m2.

Crystals in the microplates were aligned to beam position before collecting data at room temperature or cryogenic temperature. Firstly, the edge of the membrane was aligned to beam position at low magnification and then the microplate was rotated by 90◦ to locate the side of the centerline. Further steps were performed to bring the protein well to the beam and make the crystals align to beam position at high magnification. At room temperature, we used the optical method to locate lysozyme microcrystals due to high radiation damage, grid scanning was used to locate lysozyme microcrystals due to the protection of cryoprotectant at cryogenic temperature. The scanning area and the type of grid scanning were selected, BluIce system automatically scanned each grid point in the area with low-dose X-rays after setting the parameters, and then automatically calculated the initial diffraction value of each grid point and draw the diffraction pattern. Datasets were collected with a small wedge angle for each crystal.

Radiation damage is a factor that must be considered for data collection at room temperature or low temperature. X-rays will affect the life of the crystal. The life of the crystal is considered as the radiation dose that the crystal can receive without structural changes. Radiation damage is divided into overall radiation damage and local radiation damage. The overall radiation damage is not for a specific atom, it is mainly manifested in the diffraction pattern, which makes the resolution of protein crystals decrease, especially the high-resolution shell. Local radiation damage is the direct inelastic scattering of X-rays with the sample, through light absorption or Polly Compton scattering. Local radiation damage breaks some covalent bonds of protein molecules, such as disulfide bonds, which appear as a small group of atoms in the electron density map. Local radiation damage can cause researchers to misunderstand the crystal structure. The lifetime of the crystal depends on many factors, such as luminous flux density, wavelength, and protein sample composition (including molecular weight, heavy atom content). The absorption of X-ray by heavy atoms is higher than that of other atoms. Regarding the overall radiation damage, Owen et al. [22] proposed that the decrease in the diffraction quality of protein crystals produced by synchrotron radiation and the local changes in the protein structure can be measured by radiation dose. Local radiation damage depends on many factors, such as the folding of protein structures. This kind of radiation damage is difficult to judge from the diffraction quality, and can only be found by analyzing the structure. According to the radiation dose limit, we collected five diffraction images at room temperature and 40 diffraction images at cryogenic temperature. The exposure time for both was set to 0.5 s with a degree increment of 1◦.
