Micrort

X-ray data were collected using the macromolecular crystallography beamline F1 at the Cornell High Energy Synchrotron Source (CHESS), which provided a 12.693 keV X-ray beam collimated to 0.1 mm diameter. Room-temperature data collection was performed using the plastic capillary sheathing method^{48 (link)}. Crystals were harvested using low-scatter kapton loops (MicroLoops, MiTeGen), taking care to minimize the amount of solvent surrounding the crystal, and placed within 2 mm diameter, 25 μm wall poly(ethylene terephthalate) capillaries (MicroRT, MiTeGen) with 10 μL reservoir solution in the tip. During X-ray exposure, images were recorded every 0.1^∘ using a pixel-array detector (Pilatus3 6M, Dectris) while rotating the sample at 1^∘ s⁻¹. A dose rate of 1.3 kGy s⁻¹ was estimated assuming a flux of 2.5 × 10¹⁰ photons s⁻¹ and a mass energy-absorption coefficient⁴⁹ of μ_en ∕ ρ = 2.0 cm² g⁻¹. After 50 s of exposure (~65 kGy), the sample was refreshed by translating to a new spot or replacing the crystal. A background dataset was collected for each crystal by translating the sample out of the beam along the spindle axis and collecting 1 s exposures while rotating at 1^∘ s⁻¹.

As a reference, a conventional X-ray diffraction experiment that used the standard oscillation method was conducted at room temperature with a crystal taken from the same crystallization batch as for the acoustic levitation experiments. After fishing a crystal on a kapton loop, we sealed it in a plastic capillary tube with a drop of crystallization buffer to avoid dehydration of the crystal during the experiment, using a room temperature experimental kit (MicroRT, MiTeGen, Ithaca, NY, USA). The crystal was rotated by a single phi axis goniometer at an oscillation range of 0.2°, and the diffraction images were collected at an exposure time of 0.1 second at a detector distance of 300 mm. We translated the crystal by 10 μ m for every 10 degrees of rotation to minimize the radiation damage. This procedure mimics the constant renewal of the crystal region exposed to X-rays in the levitation method, due to the spinning and orbiting of the crystal within the droplet. We collected 900 diffraction images in total. The dataset obtained was indexed and processed with XDS and XSCALE42 . We then solved the crystal structure by applying the molecular replacement method using MOLREP43 and refined by using REFMAC44 in the CCP4 program suite45 . The PDB code 4XJB46 was used as the model structure for the molecular replacement. See Table 1 for the detailed processing statistics.