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| Title | Off-target structural insights: ArnA and AcrB in bacterial membrane-protein cryo-EM analysis. |
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| Journal, issue, pages | Acta Crystallogr D Struct Biol, Vol. 81, Issue Pt 10, Page 545-557, Year 2025 |
| Publish date | Oct 1, 2025 |
 Authors | Mehmet Caliseki / Ufuk Borucu / Sathish K N Yadav / Christiane Schaffitzel / Burak Veli Kabasakal /  |
| PubMed Abstract | Membrane-protein quality control in Escherichia coli involves coordinated actions of the AAA+ protease FtsH, the insertase YidC and the regulatory complex HflKC. These systems maintain proteostasis ...Membrane-protein quality control in Escherichia coli involves coordinated actions of the AAA+ protease FtsH, the insertase YidC and the regulatory complex HflKC. These systems maintain proteostasis by facilitating membrane-protein insertion, folding and degradation. To gain structural insights into a putative complex formed by FtsH and YidC, we performed single-particle cryogenic electron microscopy on detergent-solubilized membrane samples, from which FtsH and YidC were purified using Ni-NTA affinity and size-exclusion chromatography. Although SDS-PAGE analysis indicated high purity of these proteins, cryo-EM data sets unexpectedly yielded high-resolution structures of ArnA and AcrB at 4.0 and 2.9 Å resolution, respectively. ArnA is a bifunctional enzyme involved in lipid A modification and polymyxin resistance, while AcrB is a multidrug efflux transporter of the AcrAB-TolC system. ArnA and AcrB, known Ni-NTA purification contaminants, were also consistently detected by mass spectrometry in Strep-Tactin affinity-purified samples, validating their presence independently of affinity-tag selection. ArnA, which is typically cytoplasmic, was consistently found in membrane-isolated samples, indicating an association with membrane components. Only 2D class averages corresponding to the cytoplasmic AAA+ domain of FtsH were observed; neither side views of full-length FtsH nor densities corresponding to an intact FtsH-YidC complex could be identified, due to the conformational flexibility of the FtsH complex and its transient interaction with YidC, which limited particle alignment and stable classification in cryo-EM data sets. Two-dimensional class averages revealed additional particles resembling GroEL and cytochrome bo oxidase. These results underscore the utility of cryo-EM in uncovering off-target yet structurally well defined complexes, which may reflect physiologically relevant interactions or purification biases during membrane-protein overexpression. |
 External links | Acta Crystallogr D Struct Biol / PubMed:40927951 / PubMed Central |
| Methods | EM (single particle) |
| Resolution | 2.92 - 4.0 Å |
| Structure data | EMDB-64789, PDB-9v5h: EMDB-64793, PDB-9v5r: |
| Source |
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 Keywords | TRANSFERASE / Lipid A modification Polymyxin resistance Bifunctional enzyme UDP-glucuronic acid dehydrogenase UDP-4-amino-4-deoxy-L-arabinose / transferase Lipopolysaccharide biosynthesis Antibiotic resistance Aminotransferase Dehydrogenase LPS modification pathway L-Ara4N biosynthesis / TRANSPORT PROTEIN / Multidrug efflux pump / Trimeric transporter / RND transporter / Proton motive force / Active transport / Antimicrobial resistance / Drug efflux / Inner membrane transporter / AcrAB-TolC complex |
escherichia coli (E. coli)