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Pipeline publication

[…] lex at a concentration of ∼20 mg/ml, which had been supplemented with 0.01% DDM were placed onto glow-discharged holey carbon grids (Cu R 1.2/1.3 200 mesh, Quantifoil). Grids were blotted for 2.5 s and flash frozen in liquid ethane using an FEI Vitrobot. Images were taken on FEI Titan Krios electron microscope operating at 300 kV with a nominal magnification of 22 500×. Images were recorded by a K2 Summit detector (Gatan) with the super-resolution mode, and binned to a pixel size of 1.32 Å. Defocus values varied from 1.5 to 2.5 μm. Each image was dose-fractionated to 32 frames with a dose rate of ∼8.2 counts/sec/physical-pixel (∼4.7 e−/sec/Å2), total exposure time of 8 and 0.25 s per frame. UCSFImage4 was used for all data collection (developed by XL)., A total of 1 064 cryo-EM micrographs were collected. All images were aligned and summed using whole-image motion correction. The defocus value of each image was determined by CTFFIND3. Auto-picking was performed using the reference-based particle picking subroutine in RELION and the templates for particle auto-picking were obtained from the 2D class averages calculated from ∼5 000 manually picked particles. After manual check of the auto-picking results, 306 675 particles were extracted. Reference-free 2D classification was applied to remove ice and contaminants yielding 274 341 good particles. After two rounds of 3D classification, 157 243 particles were selected and 3D reconstruction with D2 symmetry was preformed resulting in a resolution of 7.3 Å. Then soft mask was performed and the reported resolution was increased to 5.4 Å. All reported resolutions are based on the gold-standard FSC = 0.143. All processions were performed in RELION., Given the avai […]

Pipeline specifications

Software tools UCSFImage, CTFFIND, RELION