K100x glow discharge unit

NS-EM sample preparation was performed on 400-mesh copper grids with carbon film (Agar Scientific). Grids were glow-discharged for 30 s at 45 mA using a K100X glow discharge unit (Electron Microscopy Sciences) before a 4-µl sample was applied to the grids and incubated for 2 min. Grids were stained by two successive applications of 4 µl 2% (w/v) uranyl acetate with blotting between the first and second application. Stained grids were blotted after 20 s to remove excess stain. Unless described otherwise, data collection was carried out on a Tecnai LaB6 G2 Spirit transmission electron microscope (FEI) operating at 120 keV. A 2K × 2K GATAN Ultrascan 100 camera was used to collect micrographs at a nominal magnification of 30,000 (with a physical pixel size of 3.45 Å per pixel) within a −0.5 to −2.0 µm defocus range.

CMG assembly reactions (reconstituted as described in ‘In vitro CMG assembly on short chromatinized origins’) were frozen on 400-mesh lacey grids with a layer of ultra-thin carbon (Agar Scientific). All grids were freshly glow-discharged for 1 min at 45 mA using a K100X glow discharge unit (Electron Microscopy Sciences) before plunge freezing. Samples were prepared by applying 4 µl of undiluted CMG assembly reactions for 2 min on a grid equilibrated to 25 °C in 90% humidity. The grid was blotted for 4.5 s and plunged into liquid ethane. Data collection was performed on an in-house Thermo Fisher Scientific Titan Krios transmission electron microscope operated at 300 kV, equipped with a Gatan K2 direct electron detector camera (Gatan) and a GIF Quantum energy filter (Gatan). Images were collected automatically using the EPU software (Thermo Fisher Scientific) in counting mode with a physical pixel size of 1.08 Å per pixel, with a total electron dose of 51.4 electrons per Ų during a total exposure time of 10 s dose-fractionated into 32 movie frames (Extended Data Table 1). We used a slit width of 20 eV on the energy filter and a defocus range of −2.0 to −4.4 μm. A total of 65,286 micrographs were collected from two separate sessions.