Off‐target structural insights: ArnA and AcrB in bacterial membrane‐protein cryo‐EM analysis

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 bo3 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.