Extended Data Fig. 1: Cor inhibits M. tuberculosis RNAP transcription initiation but not promoter DNA binding and data-processing pipeline for the cryo-EM movies of RNAP–σA–CarD–RbpA–Cor–AP3 promoter. | Nature

Extended Data Fig. 1: Cor inhibits M. tuberculosis RNAP transcription initiation but not promoter DNA binding and data-processing pipeline for the cryo-EM movies of RNAP–σA–CarD–RbpA–Cor–AP3 promoter.

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

Extended Data Fig. 1

a, Chemical structure of Cor40. b, Abortive transcription initiation assays measuring GpUpU production in the presence of increasing concentrations of Cor. 32P-labelled abortive transcript production was monitored by polyacrylamide gel electrophoresis and autoradiography. Full gel is shown in Supplementary Fig. 1. c, Flow chart showing the image-processing pipeline for the cryo-EM data of M. tuberculosis RNAP–σA–CarD–RbpA–Cor–AP3 promoter complexes, starting with 3,718 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. After picking, the dataset contained 1,026,386 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, three rounds of 3D heterogeneous refinement were performed using cryoSPARC in a binomial-like fashion. One major, high-resolution class emerged, which was refined using cryoSPARC homogenous refinement and then sharpened for model building.