*3.2. In Situ Crystal Location*

Once crystals were grown in microplate A or B, microplates were mounted on the goniometer head of the MD2 diffractometer for data collection, and it was important to quickly achieve precise alignment between the microcrystals and the incident X-rays. Unlike many other in situ data collection with traditional plates systems, our microplates can be moved into the beam position without adding any other motor systems. Moreover, crystals can be clearly focused to the beam position with the beamline centering system. Figure 3a,b shows the typical working position of microplates A and B at the beamline. However, it is extremely difficult to align the crystal to beam position with normal 96-well crystal plates, because normal sized crystal plates are difficult to rotate 90◦ to align the crystal to the beam position.

**Figure 3.** Microplates mounted on the goniometer head for data collection. (**a**) Plate A with 10 protein wells was installed on the goniometer head. (**b**) Plate B with 18 protein wells was installed on the goniometer head. Crystal location using different grid scanning types: (**c**) rectangle scanning area; (**d**) oval scanning area; (**e**) line scanning area; (**f**) polygon scanning area.

Crystal location results using grid scanning with plate B under cryogenic temperature are shown in Figure 3. Our results show that the crystal position, located on the Kapton membrane, can be easily identified by grid scanning. Four grid scanning types (rectangle, oval, line, polygon) can be used for plate B to promote the crystal location, the grid size, rotation wedge, and exposure time that can be defined by the user. The Kapton membrane does not affect the crystal location by grid scanning, moreover, to confirm that there is no effect of Kapton membrane on the crystal location, the effect of Kapton membrane is investigated in Section 3.3.

The combined use of the in situ diffraction plate and grid scan significantly improves the efficiency of diffraction data screening and collection. The common data collection of crystals requires each crystal to be separately installed on the goniometer, centered under the X-ray irradiation, and then removed and the sample changed. For this experiment, there are hundreds of lysozyme microcrystals in a protein well, and a set of plates can be loaded with thousands of crystals at the same time. Grid scanning can scan the entire protein well at once, and then it can quickly determine the position of all crystals in a protein well and the preliminary diffraction value of that position will be displayed. Click the grid point with high diffraction value, and the system will automatically align it with the optical path, which can greatly reduce the time of sample exchange and positioning.
