Room temperature imaging was performed either on a 120 keV Tecnai Spirit (Thermo Fisher) or a 200 keV Talos 200 C (Thermo Fisher) electron microscope. Datasets on the Tecnai Spirit were collected at a nominal magnification of 52,000X (2.05 Å/pix) with a Tietz TVIPS CMOS 4k x 4k camera, with a defocus of −1.5μM and a total dose of 25 e-/Å2. Datasets on the Talos were collected at a nominal magnification of 73,000X (1.98 Å/pix) with a 4k × 4k CETA camera (Thermo Fisher), with a defocus of −1.5μM and a total dose of 25 e-/Å2. Leginon44 (link) was used for automated data collection, and micrographs were stored in the Appion database45 (link). Single particle analysis was performed in RELION46 (link), including CTF estimation, using CtfFind447 (link), particle picking, and reference-free 2D classification. For 3D classification, our previous negative stain reconstruction of 311-rsCSP, low-pass filtered to 60 Å, was used as a reference. High-quality 3D classes were used as references for 3D refinement in RELION, and C1 symmetry was used in all cases.
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