Cryo-EM Structure Determination of Bacterial RNAP

The grids were imaged using a 300 kV Titan Krios (FEI, Inc.) equipped with a K2 Summit direct electron detector (Gatan, Inc.). Images were recorded with Serial EM in counting mode with a physical pixel size of 1.307 Å and a defocus range of 1.5–2.5 μm. Data were collected with a dose of 8 e/pixel/s. Images were recorded with a 12 s exposure and 0.25 s subframes to give a total dose of 59 e/Ų. Subframes were aligned and summed using MotionCor2 (22 (link)). The contrast transfer function was estimated for each summed image using CTFFIND4 (23 (link)). From the summed images, ∼10 000 particles were manually picked and subjected to 2D classification in RELION (24 (link)). 2D averages of the best classes were used as templates for auto-picking in RELION. Auto-picked particles were manually inspected, then subjected to 2D classification in RELION. Poorly populated classes were removed, resulting in a dataset of 610 066 particles. These particles were 3D classified in RELION using a map of E. coli RNAP-promoter open complex low-pass filtered to 40 Å resolution as a reference (25 (link)). 3D classification resulted in four classes. Particles in Class 1 (RPo) were 3D auto-refined and post-processed in RELION. Particles in Class 3 were subjected to an additional 3D classification and the class with density for SspA was 3D auto-refined and post-processed.