*2.1. Design for Microplates*

As shown in Figure 1, plate A and B are manufactured using a three-dimensional printer. The size of the microplate depends on the limitation of the goniometer and the spray area of the liquid nitrogen nozzle. The diameter of the spraying area of the liquid nitrogen nozzle is 6 mm at BL18U1, and the Y-axis limitation of the goniometer is ±3 cm. In order to enable the crystal samples in the microplate to be moved to the X-ray optical path, and the crystal samples to be covered by liquid nitrogen cooling gas, the size of the microplate is designed to be less than or equal to 20 mm. Plate A has five crystallization chambers, and each crystallization chamber is composed of two protein wells and a reservoir well, and the common space is designed in the crystallization chamber to support sitting vapor-diffusion crystallization experiments. Each protein well and reservoir well are hollowed out (Figure 1a), and the position of the bottom column is on the side of the protein wells to ensure that the bottom center of plate A and the bottom center of the protein wells are on a straight line (defined as the centerline). It is worth noting that the distance between any point of the bottom of protein wells and the centerline does not exceed 1 mm. The reason for this is that the deflection of plate A does not cause the crystal to deviate from the optical path during data collection. The size of the bottom column is designed to match the magnetic base to be stably fixed on it, and the size of the reservoir wells can be changed according to different crystallization conditions. Plate A is sealed by two Kapton membranes for sitting-drop vapor-diffusion crystallization experiments after loading the protein samples.

**Figure 1.** (**a**) Crystallization plate (Plate A) used for sitting-drop vapor-diffusion crystallization experiments; (**b**) Crystallization plate (Plate B) used for hanging-drop vapor-diffusion crystallization experiments; (**c**) Incubation chamber for Plate B by covering, crystallization buffers loaded into each chamber for screening of crystallization conditions for Plate B; (**d**) Incubation chamber for Plate B by insertion. (**e**) Assembly image for Plate B with incubation chamber in Figure 1c; (**f**) Assembly image for Plate B with incubation chamber in Figure 1d; (**g**) three-dimensional device structure of plate A; (**h**) three-dimensional device structure of plate B.

Plate B has six large protein wells and twelve small protein wells (Figure 1b). Incubation chambers (Figure 1c,d) are hollowed out. The hollowing of the reservoir well not only provides a preliminary observation of the crystal through the microscope but also facilitates the cleaning and secondary use of the plate. The size of the protein wells (Figure 1b) can be selected to fit different experimental settings, the bottom center of plate B and the bottom center of large protein wells are on a straight line (defined as the centerline), and the distance between any point of the bottom of the protein wells and the centerline does not exceed 1 mm. Unlike plate A, plate B utilizes a specially designed crystallization plate in the crystallization experiment. The Kapton membrane only needs to seal the side where the centerline is. After loading the protein samples onto plate B, then plate B was assembled with the incubation chambers (Figure 1c,d) for hanging-drop vapor-diffusion crystallization experiments (Figure 1e,f). There are six reservoir wells in the incubation chambers (Figure 1c,d), which is sealed by the Kapton membrane at the bottom side before loading the crystallization buffers into each well.
