Extended Data Fig. 8: Cryo-EM of RNAP–σA–RbpA–Cor–us-fork. | Nature

Extended Data Fig. 8: Cryo-EM of RNAP–σA–RbpA–Cor–us-fork.

From: Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding

Extended Data Fig. 8

a, Flow chart showing the image-processing pipeline for the cryo-EM data of RNAP–σA–RbpA–Cor–us-fork complexes, starting with 4,897 dose-fractionated movies collected on a 300-keV Titan Krios (FEI) equipped with a K2 Summit direct electron detector (Gatan). Movies were frame-aligned and summed using MotionCor229. CTF estimation for each micrograph was calculated with Gctf30. A representative micrograph is shown following processing by MotionCor2. Particles were autopicked from each micrograph with Gautomatch and then sorted by 2D classification using RELION33 to assess quality. The twelve highest-populated classes from the 2D classification are shown. The dataset contained 925,009 particles. A subset of particles was used to generate an ab initio map in cryoSPARC31. Using the low-pass-filtered (30 Å) ab initio map as a template, two rounds of 3D heterogeneous refinement were performed using cryoSPARC in a binomial-like fashion. One major class emerged, which was refined using cryoSPARC homogenous refinement and then sharpened for model building. b, Angular distribution calculated in cryoSPARC for particle projections. Heat map shows number of particles for each viewing angle. c, Gold-standard FSC41, calculated by comparing the two independently determined half maps from cryoSPARC. The dotted line represents the 0.143 FSC cut-off, which indicates a nominal resolution of 4.4 Å. d, RNAP clamp conformations. The RPo structure (Fig. 1c) was used as a reference to superimpose other structures via α-carbon atoms of the structural core module (Supplementary Table 2), revealing a common core RNAP structure (grey molecular surface) but with shifts in the clamp modules. The clamp modules are shown as backbone cartoons with cylindrical helices (RPo, blue; Cor–RbpA-holo, olive; RbpA-holo (6C05), cyan; open-clamp (6BZO), red). The angles of clamp opening are shown (relative to RPo at 0°).