K3 summit

For the full-length IrtAB, IrtA_H407AB_ΔSID (E-Q) and IrtA_H407DB_ΔSID (E-Q) samples, cryo-EM data were collected on a 300 kV FEI Titan Krios electron microscope with a Gatan K3 Summit direct electron detector at a nominal magnification of 29,000 with a pixel size of 0.82 Å in super-resolution mode. Movies were recorded for 2.4 s in 40 sub-frames with a total dose of 60 e⁻/Ų. For IrtAB_ΔSID with 1 mmol/L AMP-PNP-Mg²⁺, IrtAB_ΔSID with 5 mmol/L ADP-Mg²⁺ and IrtAB_ΔSID (E-Q) with 10 mmol/L ATP-Mg²⁺ samples, a Gatan K2 Summit direct electron detector, equipped with a Gatan quantum energy filter, was used to capture movies at EFTEM magnification of 165,000 in super-resolution mode with a pixel size of 0.82 Å. For IrtAB_ΔSID with 10 mmol/L AMP-PNP-Mg²⁺ and IrtAB (E-Q) with 10 mmol/L ATP-Mg²⁺ samples, a Gatan K3 Summit direct electron detector, equipped with a Gatan quantum energy filter, was used to capture movies at EFTEM magnification of 105,000 in super-resolution mode with a pixel size of 0.832 Å.

Cryo-EM data was collected on a Titan Krios transmission electron microscope operating at 300 keV and micrographs were acquired using a Gatan K3 summit direct electron detector. The total dose was 60 e^-/Ų, fractioned over 100 frames during a 10 s exposure. Data was collected at 105,000× nominal magnification (0.832 Å/pixel at the specimen level) and nominal defocus range of –1.0 to –2.0 µm.

We used a Vitrobot Mark IV (Thermo Fisher) to prepare cryo-EM grids of the various Pol α-primase samples. A 3-μl droplet of the samples at a final concentration of ~1 mg/ml was pipetted onto C-flat 2/1 holey carbon grids, treated by glow-discharge immediately before use. The grids were then incubated for 10 s at 6 °C in 100% humidity, blotted for 3 s, and then plunged into liquid ethane. EM grids were loaded into a Titian Krios electron microscope operated at 300 kV, and images were collected automatically in low-dose mode at a magnification of ×130,000 with an objective lens defocus ranging from −1.5 to −2.5 μm. A Gatan K3 Summit direct electron detector was used for image recording in the super-resolution mode with a pixel size of 0.828 Å at the sample level. The dose rate was 10 electrons per Ų per second and the total exposure time was 6 s. The total dose per micrograph was divided into a 30-frame movie, so each frame was exposed for 0.2 s. We used SerialEM^{46 (link)} to collect one dataset of 10,519 raw movie micrographs for Pol α alone in the apo form, one dataset of 3357 raw movie micrographs for Pol α-T complex, one dataset of 14,691 micrographs for the Pol α–T/P8 complex, one dataset of 13,433 micrographs for the Pol α–T/P10 complex, and one dataset of 17,525 micrographs for the Pol α–T/P15 complex.