1. ABOUT THE DATASET -------------------- Title: BAM-BepA complexes in outer membrane protein quality control Creator(s): Katherine L. Fenn, Victoria Higgins, Jonathan M. Machin, Antonio N. Calabrese, Alan Berry, Sheena E. Radford†, Neil A. Ranson† Organisation(s): Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK Rights-holder(s):Unless otherwise stated, Copyright 2026 University of Leeds Publication Year: 2026 Description: Correct folding of outer membrane proteins (OMPs) by the β-barrel assembly machinery (BAM) is essential for maintaining the outer membrane (OM) barrier function of sidearm bacteria. When OMP biogenesis is perburbed, the β-barrel assembly enhancing protease A(BepA) binds to BAM to mediate quality control but how BepA interacts with BAM and degrades substrate OMPs remains unclear. Here we present, AF3 predictions of BepA, BepA with BAM from 11 bacterial species, BepA with 15 residues peptides encompassing the entire sequence of 3 OMPs (BamA, LptD, OmpX) and HPLC data from BepA and BepA mutants degrading a series of OMP peptides. Cite as: Katherine L. Fenn, Victoria Higgins, Jonathan M. Machin, Antonio N. Calabrese, Alan Berry, Sheena E. Radford†, Neil A. Ranson†. (2026): Data for ' BAM-BepA complexes in outer membrane protein quality control' University of Leeds https://doi.org/10.5518/1838 Related publication: [Provide a citation for any article reporting results based on analysis of the dataset. Direct links to related publications can also be added to the Related resources fields in the metadata record. If a publication is in preparation at the time of deposit, provide relevant details where known (authors, title, journal, year, etc.) and indicate status at time of deposit (Submitted; In preparation; Accepted).] Contact: fbskfe@leeds.ac.uk n.a.ranson@leeds.ac.uk 2. TERMS OF USE --------------- Copyright 2026 University of Leeds. Unless otherwise stated, this dataset is licensed under a Creative Commons Attribution 4.0 International Licence: https://creativecommons.org/licenses/by/4.0/. 3. PROJECT AND FUNDING INFORMATION ---------------------------------- K.L.F. and V.H. are funded by the BBSRC (BB/X015653/1). JMM is funded by MRC (MR/Y012453/1). SER holds a Royal Society Professorial Fellowship (RSRP/R1/211057). 4. CONTENTS ----------- AF3_BAM_BepA_otherspecies - contains AF3 models and confidences of BAM with BepA from 10 species AF3_BepA - contains AF3 models and confidences of app-BepA AF3_BepA_peptides - contains 350 AF3 predictions of peptides with BepA(Δlid) each with 5 models. AF3_OtherPlugEnzymes - contains AF3 models and confidences for YcaL, LoiP, HptX and Oma1. HPLC_rawdata - contains raw HPLC traces for BepA and BepA mutants in the presence of different OMP peptides AF3_BAM_BepA.pdb - AF3 model of BAM BepA from E.coli with Lid engaged BepA. 5. METHODS ---------- Alphafold3 models of BAM-BepA from diderm WHO priority pathogens Proteins were identified by sequence and structural homology compared to their E. coli counterparts. The highest priority species from the WHO priority pathogen list5 was chosen where multiple genera occurred. Where a specific species was not indicated the most common species was utilised. Models generated using the AlphFold server. AF3 models of BepA and other plug enzymes were also generated using the AF3 server. AF3 BepA(Δlid) with 15mer peptides AF3 installed on a local workstation was used to predict BepA(Δ1-40, 150-199) (the lid and N-terminus were removed for all predictions) in the presence of Zn2+, H2O and 15 residue peptides. The 15 residues peptides encompassed the whole sequence of LptD, BamA and OmpX moving along each sequence by 5 residues totalling 350 peptides. 5 models were predicted per peptide. For each model, the distances from the zinc ion to both NE2 and ND1 of the regulatory histidine were calculated using Biopython61. Models where both distances were greater than 3Å were designated plug-out conformation In plug out models, peptide interactions with the exposed edge strand were identified using a Cα–Cα distance-based contact analysis, where peptides containing residues within 5 Å of residues 64–69 (corresponding to residues 105-110 in BepA sequence) were classified as interacting. β-strand formation was assessed using DSSP secondary structure assignment (mkdssp62, Sali lab implementation), accessed via Biopython. Strand orientation was determined from the relative direction of backbone Cα vectors, allowing classification as parallel or antiparallel. For each plug-out model, the residue closest to the valine in the pocket were identified using Biopython (Val-92 in AF3 predictions corresponding to Val-133 in BepA sequence). The composition of residues in this pocket was analysed to determine enrichment relative to the background sequence. For each amino acid, enrichment was calculated as the log2 ratio of the observed frequency to the background frequency. Residues were then grouped into chemical class and a sequence window +/- 5 residues around the identified residue was extracted. Enrichment was calculated as a log2 ratio of observed to background frequencies and visualised as a heatmap. OMP peptide proteolysis analysed via HPLC Peptides were solubilised in 100% DMSO at >1mM and diluted to 100μΜ in 20mM Tris-HCl pH8, 150mM NaCl. 10μM peptide was added to 3μΜ ΒepA (or mutant). The reaction was stopped at a given time point (0, 2, 5, 10, 20, 30, 45, 60, 90, 120, 180, 240 or 300 minutes) using 10mM 1,10 phenanthroline (Merck). The samples were analysed by Reverse-phase HPLC on a Shimadzu Nexera LC-40 system. 10μl of sample was injected onto a C8 column (Phenomenex) using a linear gradient from 5-95% acetonitrile in 0.1% TFA over 20 minutes with detection of tyrosine fluorescence (Ex 275nm, Em 305nm). QMN peptide = QMNKMGGFNLKYRYE7, GVI peptide = GVIGSFTYTEKSRTA7, KHD peptide = KHDTSDYGFSYGAGL7 , ARY = QMNKMGGFNLKARYE7, YRA = QMNKMGGFNLKYRAE7 For samples comparing the QMN peptide mutation (QMN, ARY, YRA), samples were analysed in the absence of phenanthroline.