The biogenesis of β-barrel outer membrane proteins is mediated by the β-barrel assembly machinery (BAM) in Gram-negative bacteria. In E. coli, BAM is a heterooligomeric complex composed of the central β-barrel protein BamA and associated lipoproteins BamB-E [1]. BamA is a highly conserved and essential protein, making it an attractive target for novel antibiotics. BamA and BamD are the only essential components of the system and the role of lipoproteins or how the conformational dynamics of the complex is regulated remains elusive. We used site-directed spin labeling (SDSL) combined with pulsed electron-electron double resonance (PELDOR/DEER) spectroscopy to elucidate the function of lipoproteins in vitro and to observe the conformational state of the machinery in intact E.coli [2]. We introduced cysteine pairs at the lateral gate, extracellular loops, and periplasmic regions of the core component BamA. The cysteine pairs were spin labeled in vitro in different oligomeric states of the machinery and the distances were experimentally determined. In detergent micelles, BamAB is observed like BamA [3] exclusively in an inward-open state characterized mainly by the closure of the lateral gate region of the barrel. However, BamACDE and BamABCDE complexes show an opening on the lateral gate region. The presence of BamCDE subcomplex also increased the overall conformational heterogeneity of BamA. The extracellular loops L6 and L8 exist in a dynamic state spanning several conformations as observed from available structures. Interestingly, a segment of L6 displays a highly dynamic behaviour irrespective of the lipoproteins present. Additional experiments in intact E. coli cells further confirmed the heterogeneity of BamABCDE in the cellular environment. In summary, our results reveal that the accessory lipoproteins regulate the conformational cycling of BamA.