(A) Cryo-EM density maps and superimposed refined models for Mtb RbpA/σA-holo (5.2 Å resolution) and Mtb Fdx/RbpA/σA-holo (6.5 Å resolution). (B) RNAP clamp conformational changes for four cryo-EM structures determined in this work. The RbpA/RPo (Figure 2A) structure was used as a reference to superimpose the other structures via α-carbon atoms of the structural core module (Supplementary file 2), revealing a common core RNAP structure (shown as a gray molecular surface) but with large shifts in the clamp modules. The clamp modules are shown as backbone cartoons with cylindrical helices and color-coded (blue, closed clamp of RPo; green, relaxed clamp of RbpA/σAholo; orange, open clamp of Fdx/RbpA/σA-holo/us-fork; red, open clamp of Fdx/RbpA/σA-holo). The clamp conformational changes can be characterized as rigid body rotations about a rotation axis perpendicular to the page (denoted). The angles of clamp opening for the different structures are shown (relative to the blue closed RPo clamp, 0° opening). (C) The core RNAP from the 3.4 Å resolution Fdx/RbpA/σA-holo/us-fork structure is shown as a gray molecular surface but with the open clamp colored orange. The structure is sliced at the level of the Fdx binding pocket (the bound Fdx is shown in green). The boxed region is magnified below, showing the tight fit of the Fdx molecule in a narrow gap between the clamp and the rest of the RNAP. (D) The core RNAP from the 3.3 Å resolution RbpA/RPo structure is shown as a gray molecular surface but with the closed clamp colored blue. The structure is sliced at the level of the (empty) Fdx binding pocket. Fdx, modeled from the structure shown in (C), is shown in green. The boxed region is magnified below. Fdx cannot bind to RNAP with a closed clamp because clamp closure pinches off the Fdx binding site. Clamp closure is required for initiation and stabilization of the transcription bubble (Feklistov et al., 2017) and also for stable binding of nucleic acids in the RNAP cleft.