Fig. 3: BcsF-dependent BcsE recruitment and regulatory complex conformation in the c-di-GMP-saturated state.

a Locally refined cryo-EM structure of the BcsE₂F₂ assembly from the c-di-GMP-saturated synthase macrocomplex shown as electron density and in cartoon. IM, inner membrane. b BcsF dimerization shown as Coulombic electrostatic potential-colored surface (left, default −10 to 10 range) and in cartoon and sticks (right). c BcsE-BcsF interactions. Left, BcsE^NTD is shown as a lipophilicity-colored surface (default −20 to 20 range), BcsF residues—including the hydrophobic plug residues V⁴⁶ and L⁵²—are shown as sticks. Right, recombinant expression and purification of the Bcs macrocomplex with various BcsF variants (Bcs^HisRQA^HA-FLAGB + Bcs^strepEF*G). Protein-specific bands are identified as previously^17,21. BcsE and BcsA-specific signals are further detected by western blotting with epitope tag-specific antibodies in the bottom (representative data from three independent experiments). d The BcsE^NTD dimerization interface. e The BcsE^REC* dimerization interface. f The c-di-GMP-binding dual I-site pocket in closed BcsE. All interface parameters were calculated with PISA⁵⁷.