Extended Data Fig. 1: Construct design and purification of Sgl–MurJ complexes.
From: Convergent MurJ flippase inhibition by phage lysis proteins
a, Cartoon representation of TaMurJ crystal structures in inward closed (PDB:6NC6) and outward (PDB: 6NC9) states and the EcMurJ crystal structure (PDB:6CC4) in an inward closed state with the N-lobe, C-lobe, and TMs 13-14 colored (blue, green and pink, respectively). b, Crystal structure of EcMurJ BRIL fusion (EcMurJBRIL) in an inward closed state showing the full construct. The distortion of TM1 and the deleted C-terminal residues are highlighted by dashed boxes. c, Left top, schematic of co-expression constructs for the Sgls and MurJ. Left bottom, various constructs of EcMurJ in a MurJ-depletion strain. The genomic EcMurJ is controlled by an arabinose promoter. In the absence of arabinose, only cells expressing a complementing MurJ are viable, in this case under the control of an IPTG inducible promoter. The construct used for structural studies, EcMurJBRIL, complements similar to wild-type MurJ. The non-functional R24A mutant33 is included as a negative control. Right, Lysis assay comparison of the His-tagged SglM in the absence and presence of our co-expressed EcMurJBRIL, demonstrating that EcMurJBRIL can rescue lysis. d, Representative size exclusion chromatography profiles and SDS-PAGE analyses of purified Sgl–MurJ and Sgl–MurJ–Fab–Nb complexes. Molecular weight markers are indicated in KDa. The SglCJ3 band was not observed on the SDS-PAGE gel, likely due to low sensitivity. The predicted location of SglCJ3 is labeled.
