XFEL data of CysLT1R-zafir crystals were collected at the CXI (coherent x-ray imaging) instrument of the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory, Menlo Park, California. The LCLS was operated at a wavelength of 1.302 Å (9.52 keV) delivering individual x-ray pulses of 43-fs duration focused into a spot size of ~1.5 μm in diameter using a pair of Kirkpatrick-Baez mirrors. LCP laden with protein microcrystals was injected at RT inside a vacuum chamber into the beam focus region using an LCP injector (42) with a 50-μm-diameter capillary at a flow rate of 0.3 μl min−1. Microcrystals ranged in size from 2 to 10 μm. Diffraction data were collected at a pulse repetition rate of 120 Hz with the 2.3-megapixel Cornell-SLAC Pixel Array Detector. The beam was attenuated to 6.3% of full intensity (1.9 × 1010 photons per pulse) to avoid detector saturation. A total number of 315,374 detector images were collected, of which 40,720 were identified as potential crystal hits with more than 20 Bragg peaks using Cheetah (43), corresponding to an average hit rate of 13%. Autoindexing and structure factor integration of the crystal hits were performed using the Monte Carlo integration routine with the “pushres 2.4” option implemented in CrystFEL (version 0.6.3 + 23ea03c7) (44). Peak detection parameters were extensively optimized for Cheetah, and experimental geometry was refined for CrystFEL with geoptimizer (45). The overall time of data collection from a sample with a total volume of about 15 μl was approximately 45 min and yielded 30,129 indexed patterns (indexing rate 90%).

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