The rsCSP-Fab667 and rsCSP-Fab668 complexes were diluted to 0.01mg/ml using 1X TBS pH 7.4 and deposited on glow discharged carbon-coated copper-mesh grids. 2% uranyl formate was used to negatively stain the complexes for 50 seconds. Grids were transferred to a Thermo Fisher Tecnai Spirit T12 (120 keV) equipped with a TVIPS CMOS 4k x 4k camera for data collection. The Leginon software [47 (link)] was used for automated data collection and resulting images were stored in the Appion database [48 (link)]. All images were collected at 52,000 x magnification, a defocus value of -1.5 um, a dose of 25e-/Å2, and a pixel size of 2.05.
A tilted dataset was collected for rsCSP-Fab667 and rsCSP-Fab668 complexes by changing the stage angle to 0°, -10°, -20°, -30°, -40°, and -50°, resulting in 251 and 505 images, respectively. Particles were picked using DogPicker [49 (link)] and stacked with a box size of 160 or 192 pixels. Micrographs were CTF corrected using GCTF [50 (link)] and particles were extracted. The particle stack was imported into cryoSparc2 [51 (link)], where rounds of reference-free 2D classifications, initial model-free 3D classifications and 3D refinements were performed. 3D refinements did not have symmetry imposed. Crystal structures of Fab667 and 668 were fit to their respective nsEM maps using UCSF Chimera [52 (link)].
A tilted dataset was collected for rsCSP-Fab667 and rsCSP-Fab668 complexes by changing the stage angle to 0°, -10°, -20°, -30°, -40°, and -50°, resulting in 251 and 505 images, respectively. Particles were picked using DogPicker [49 (link)] and stacked with a box size of 160 or 192 pixels. Micrographs were CTF corrected using GCTF [50 (link)] and particles were extracted. The particle stack was imported into cryoSparc2 [51 (link)], where rounds of reference-free 2D classifications, initial model-free 3D classifications and 3D refinements were performed. 3D refinements did not have symmetry imposed. Crystal structures of Fab667 and 668 were fit to their respective nsEM maps using UCSF Chimera [52 (link)].