9V5R

cryo-EM structure of trimeric AcrB

Summary for 9V5R

• Entry DOI: 10.2210/pdb9v5r/pdb
• EMDB information: 64793
• Descriptor: Multidrug efflux pump subunit AcrB (1 entity in total)
• Functional Keywords: multidrug efflux pump, trimeric transporter, rnd transporter, proton motive force, active transport, antimicrobial resistance, drug efflux, inner membrane transporter, acrab-tolc complex, transport protein
• Biological source: Escherichia coli
• Total number of polymer chains: 3
• Total formula weight: 335469.77

Authors

Caliseki, M.,Borucu, U.,Kadapalakere, S.Y.,Schaffitzel, C.,Kabasakal, B.V. (deposition date: 2025-05-26, release date: 2025-07-30, Last modification date: 2025-10-08)

Primary citation

Caliseki, M.,Borucu, U.,Yadav, S.K.N.,Schaffitzel, C.,Kabasakal, B.V.
Off-target structural insights: ArnA and AcrB in bacterial membrane-protein cryo-EM analysis.
Acta Crystallogr D Struct Biol, 81:545-557, 2025

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 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.

PubMed: 40927951
DOI: 10.1107/S2059798325007089

Experimental method

ELECTRON MICROSCOPY (2.92 Å)