*3.4. Structure Determination with Microcrystals*

We used lysozyme microcrystals (less than 20 μm) to verify the in situ microplates, and the data was collected at the BL18U beamline station of SSRF. When performing the experiments at room temperature, we used the visual method (Figure 2) to select 20 crystals from plate A for diffraction data collection. According to the radiation dose limit, five diffraction images were collected for each crystal (typical pattern in Figure 5b), and the relatively poor diffraction images were deleted, then each set of data was indexed with XDS. In the end, we selected five data sets with a total of 25 images, and these images were integrated through the blend program and then used for further structural analysis. According to the above-mentioned highest resolution criteria, we finally got the highest resolution of 2.15 Å. When performing the experiments at cryogenic temperatures, we used grid scanning (Figure 3) to select 10 crystals from plate B for diffraction data collection. A total of 40 diffraction images were collected from each crystal (typical pattern in Figure 5c). The data processing is the same as above. Finally, four data sets with 40 images were integrated through the blend program and then we got the highest resolution of 1.98 Å.

**Figure 5.** Typical diffraction pattern collected from crystal mounted by Nylon loop at 100 K (**a**) and the microplate A at room temperature (**b**) and the microplate B at 100 K (**c**).

In order to compare with the common method, the nylon loop was used for diffraction data collection. We used the same method to cultivate lysozyme microcrystals, and then the same data acquisition strategy was used to collect data from lysozyme at cryogenic temperature. According to the aforementioned data processing method, we finally integrated 60 diffraction images (typical pattern in Figure 5a) and obtained the highest resolution of 1.96 Å. The comparison of data collection through the in situ microplates and the data collection through the nylon loop is shown in Table 1.


Redundancy 4.5(3.9) 3.6(2.9) 2.5(3.1)

**Table 1.** Statistical analysis of lysozyme using a nylon loop and the in situ plate for data collection. The data collection for multiple crystals was performed at a low temperature (100 K) and room

In this experiment, we developed an efficient method for sample delivery and data collection for multicrystals. A Kapton membrane was utilized for crystal growth and sealing up the microplate. A complete dataset can be obtained after merging multiple datasets and the structure can be solved. Comparing the in situ microplates with the single nylon loop, the data collected using the in situ microplates at cryogenic temperature have the same good quality as the data collected using a single nylon loop, but the data collected using the in situ microplates at room temperature demonstrate worse resolution and signal-to-noise ratio, which is because the quality of crystals is affected at room temperature (Table 1). Moreover, electronic density comparation shows that there is no significant difference between results from nylon loop at 100 K and those from microplate B at 100 K. However, we do observe that there is a slight disappearance of electronic density obtained from microplate A at room temperature (F38 blue), compared with those from the nylon loop at 100 K (F38 green) or microplate B at 100 K (F38 red) (Figure 6). Our signal-to-noise analysis and electronic density analysis show that the quality of crystals is affected by radiation damage at room temperature. However, the discrepancy does not significantly affect the results.

**Figure 6.** Enlargement of the three typical lysozyme residues and view of the extra electron density observed after partial refinement using the data set collected from crystal mounted by the Nylon loop at 100 K (green) and microplate A at room temperature (blue) and microplate B at 100 K (red). (**a**) View of Lysozyme W28 residue. (**b**) View of Lysozyme F38 residue. (**c**) View of Lysozyme Y57 residue.

In summary, this new method based on microplates realizes the in situ growth and simultaneous sample loading of multiple crystals, and it also realizes the rapid localization of crystals and the efficient data collection. On the premise that the in situ device has little effect on the data quality and low background scattering, we obtained comparable data quality to that of the traditional method with the nylon loop.

The in situ microplate including plate A and plate B has three advantages compared to the common commercially available in situ plate. First of all, except for the side parts of the microplate, the in situ microplate can almost perform a 360-degree data collection on the crystal without deviating from the optical path, and it can also be rotated at a full angle for rapid centering. The second is that the microplates are easy to manufacture and operate. The manual loading operation of the in situ microplate is the same as that of the ordinary nylon loop, which means that extra motors are not required, and it will not be limited by extra motors in most cases. The high-resolution structure of the protein can be obtained, and the manufacture of the in situ plate is fully customizable, which is suitable for most beamline stations. Third, the in situ microplates can support the cultivation of one protein under multiple crystallization conditions and the in situ cultivation of multiple proteins. This is not only suitable for the integration of multiple data sets of microcrystals, but also for multiple sets of data collection for multiple large crystals. Our microplate can save time for changing samples, and has the function of screening crystallization conditions.

However, the in situ microplates also have the following problems. When the in situ microplates are used to screen protein crystallization conditions, few crystallization conditions can be screened at a time, and it is only suitable for the fine screening of protein crystallization conditions. The size of these in situ microplates is affected by the limitation of the motor of the SSRF beamline BL18U1. In order to reduce costs, we use low-precision 3D printing technology and choose white resin materials, so far, we manually add protein samples to each well, therefore, the size of the protein wells are larger

than chip device, the distance between the two protein wells is also longer than chip device. If the researcher needs to screen more protein crystallization conditions, the in situ microplates can also be designed to have more protein wells and crystallization chambers by adopting mechanical spotting or using higher-precision processing technology, but the design of the in situ microplates needs to meet requirements mentioned in the previous section.

Furthermore, the automation level of the fixed target serial crystallography method based on the thin film in this research is far from enough, and the automatic sample delivery and data collection of crystals is still not fully automatic. In the future, the automatic data collection of crystals should be further improved. At the same time, these methods have not yet resolved protein crystals of unknown structure. These methods should be used to further test protein crystals of unknown structure to improve these methods.
