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. 2022 Sep 5;13:5226. doi: 10.1038/s41467-022-32597-2

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a SDS-PAGE of pulldown experiments examining A. aeolicus LPS binding against different CBD mutant constructs. Top and bottom panels are silver and Coomassie-blue stained gels, respectively. Uncropped images are provided as Source Data. b ATP hydrolytic activity of WzmWzt and O antigen binding mutants determined in nanodiscs and DDM micelle. WzmWzt ATPase activity measuring the maximum velocity among DDM-solubilized transporter and nanodisc reconstituted constructs in the presence (LPS) or absence (Basal) of saturating A. aeolicus LPS. Data presents the maximum velocity measured from Michaelis Menten non-linear fit model. Each data point is the means from 3 technical repeats. Error bars represent the upper and lower 95% confidence limits from the Michaelis-Menten non-linear fit (profile likelihood) c WzmWzt ATPase activity in a DDM micelle titrating 3-O-methyl-mannose, D-mannose, LPS, and O antigen. Data points represent averaged activities from three replicas normalized to the activity in the absence of substrate. Error bars represent the deviations from the means. d As in panel C but using nanodisc reconstituted WzmWzt. Individual data points are provided as Source Data. e Structure of WzmWzt in a DDM micelle shown as a surface colored according to the individual domains. Data points and error bars as stated under panel (c) f Superimposition of channel-forming nucleotide-free conformations of WzmWzt. The full-length transporter is shown in gray, the CBD-truncated construct (PDB: 6OIH) is colored according to its domains. g TM helix 1 adopts different conformations in CBD-truncated (magenta) and full-length (gray) apo WzmWzt. The structures were aligned on one Wzm subunit and the other domains are not shown for clarity. h Interactions between NBD2 and CBD2-i in the in surfo full-length WzmWzt structure.