4k 4k ceta camera

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^-/Ų. 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^-/Ų. Leginon^{44 (link)} was used for automated data collection, and micrographs were stored in the Appion database^{45 (link)}. Single particle analysis was performed in RELION^{46 (link)}, including CTF estimation, using CtfFind4^{47 (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.

For room-temperature transmission electron microscopy (TEM), cells were fixed for 2 h with a pre-warmed solution comprising 2.5% glutaraldehyde and 1.4% sucrose (pH 7.2); then, they were processed for epoxy resin embedding, as detailed by the manufacturer (Agar100 Resin Kit, Agar Scientific, Essex, UK) and as described in previous work from this group [21 (link)]. Sectioning was performed on a Leica EM UC7 ultramicrotome (Leica, Wetzlar, Germany), and the ultra-thin sections (40–60 nm) were mounted on carbon and Formvar-coated 50-mesh copper grids and counterstained with 1% uranyl acetate and Reynolds lead citrate (Agar Scientific, Essex, UK). Imaging was done using a 200 kV Talos F200C TEM, equipped with a 4K × 4K Ceta camera (Thermo Fisher Scientific, Waltham, MA, USA).