9V5H

cryo-EM structure of hexameric ArnA

9V5H の概要

• エントリーDOI: 10.2210/pdb9v5h/pdb
• 関連するPDBエントリー: 1Z7E 4WKG 6PIH
• EMDBエントリー: 64789
• 分子名称: Bifunctional polymyxin resistance protein ArnA (2 entities in total)
• 機能のキーワード: 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, transferase
• 由来する生物種: Escherichia coli; 詳細
• タンパク質・核酸の鎖数: 12
• 化学式量合計: 434769.79

構造登録者

Caliseki, M.,Borucu, U.,Kadapalakere, S.Y.,Schaffitzel, C.,Kabasakal, B.V. (登録日: 2025-05-26, 公開日: 2025-07-30, 最終更新日: 2025-10-08)

主引用文献

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

実験手法

ELECTRON MICROSCOPY (4 Å)