Journal:Acta Cryst D:S2059798325007089

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Hexameric structure of Arna (PDB-ID 5v5h), colored by the six different chains.

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Off-Target Structural Insights: ArnA and AcrB in Bacterial Membrane Protein Cryo-EM Analysis

Mehmet Caliseki, Ufuk Borucu, Sathish K. N. Yadav, Christiane Schaffitzel and Burak Veli Kabasakal ^[1]

Molecular Tour
When cells build and maintain their membranes, they need a balance of protein insertion, folding, and degradation. In Escherichia coli, this process is hypothetic to involve the AAA+ protease FtsH, the insertase YidC, and the regulatory HflKC complex. Their interaction had not been shown at structural level. To test this idea, we used single-particle cryo-electron microscopy on detergent-solubilized samples enriched for these proteins.

The results were unexpected. Instead of clear views of an FtsH–HflKC–YidC assembly, the datasets revealed , an enzyme linked to polymyxin resistance, and , the multidrug efflux transporter of the AcrAB–TolC system. Both proteins are known to appear during affinity purification, but their repeated presence across different methods and even in membrane fractions suggests that their recovery was not purely accidental. ArnA, usually described as cytoplasmic, was consistently found in membrane-enriched samples, while AcrB is a well-established membrane protein. We also observed class averages resembling GroEL and cytochrome bo3 oxidase.

These findings show that cryo-EM can capture not only the intended targets but also unexpected complexes that are well resolved and may have physiological importance. While only partial densities of the FtsH AAA+ domain were visible and no stable FtsH–YidC–HflKC complex could be reconstructed, the high-quality ArnA and AcrB structures provide fresh insights into bacterial survival strategies, from antibiotic resistance to drug efflux. More broadly, this study illustrates how structural biology can reveal unplanned discoveries that enrich our understanding of cell biology and the challenges of protein purification.

Cryo-EM density map and model fitting of the hexameric ArnA complex. (a) ArnA hexamer reconstructed at 4.0 Å resolution, shown from three orientations: top, side, and bottom views. The map reveals the characteristic two-layered architecture of ArnA and clearly resolved secondary-structure elements. (b) Final atomic model of ArnA fitted into the cryo-EM density map. Each of the 12 subunits is displayed in a different color to illustrate the hexameric arrangement. (c) Structural alignment of the final cryo-EM model (blue) (PDB ID 9y5h) with the reference crystal structure (yellow) (PDB ID 6pih) using ChimeraX. The alignment yielded an rmsd of 1.2 Å. Enlarged views show local conformational deviations in loop regions: Glu69–Ala98 in chain D (left) and Pro65–Ser75 in chain F (right). (d) Close-up view showing the model-to-map fit for a peptide segment (Leu483–Arg460). The density mesh is contoured at 1.7σ , showing clear peptide backbone density and supporting accurate model placement. Cryo-EM density map and model fitting of the AcrB trimer. (a) AcrB reconstructed at 2.92 Å resolution, shown from top, side, and bottom views. It reveals secondary-structure elements and transmembrane helices, consistent with the known trimeric architecture. (b) Final atomic model of AcrB fitted into the cryo-EM density map. Each protomer is shown in a different color to highlight the asymmetric trimer organization. (c) Structural alignment of the final cryo-EM model (blue)(PDB-ID 9v5r) with the reference crystal structure , (yellow) (PDB ID 7rr7), performed using ChimeraX. The rmsd between the structures is 1.2 Å. Enlarged views highlight conformational deviations, including E693–D711 in protomer B (left) and A704–D711 in protomer C (right), located within the PN2 subdomain. (d) Close-up view showing the density fit for side chains (W187, chain A, F572, chain B, W772. chain C). The mesh is contoured at 1.6σ in Coot, showing well-defined density for aromatic residues.

References

↑ doi: https://dx.doi.org/10.1107/S2059798325007089

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-| [[Image:022_Fig2.jpg|thumb|left|250px|Cryo-EM density map and model fitting of the hexameric ArnA complex. (a) ArnA hexamer reconstructed at 4.0 Å resolution, shown from three orientations: top, side, and bottom views. The map reveals the characteristic two-layered architecture of ArnA and clearly resolved secondary-structure elements. (b) Final atomic model of ArnA  fitted into the cryo-EM density map. Each of the 12 subunits is displayed in a different color to illustrate the hexameric arrangement. (c) Structural alignment of the final cryo-EM model (blue) (PDB ID [[9y5h]]) with the reference crystal structure (yellow) (PDB ID [[6pih]]) using ChimeraX. The alignment yielded an rmsd of 1.2 Å. Enlarged views show local conformational deviations in loop regions: Glu69–Ala98 in chain D (left) and Pro65–Ser75 in chain F (right). (d) Close-up view showing the model-to-map fit for a peptide segment (Leu483–Arg460). The density mesh is contoured at 1.7 , showing clear peptide backbone density and supporting accurate model placement.]]   [[Image:022_Fig3.jpg|thumb|right|210px|Cryo-EM density map and model fitting of the AcrB trimer. (a) AcrB reconstructed at 2.92 Å resolution, shown from top, side, and bottom views. It reveals secondary-structure elements and transmembrane helices, consistent with the known trimeric architecture. (b) Final atomic model of AcrB (PDB-ID [[9v5r]]) fitted into the cryo-EM density map. Each protomer is shown in a different color to highlight the asymmetric trimer organization. (c) Structural alignment of the final cryo-EM model (blue) (PDB ID [[7rr7]] with the reference crystal structure , yellow), performed using ChimeraX. The rmsd between the two structures is 1.2 Å. Enlarged views highlight local conformational deviations, including E693–D711 in protomer B (left) and A704–D711 in protomer C (right), located within the PN2 subdomain. (d) Close-up view showing the density fit for selected side chains (W187 in chain A, F572 in chain B, W772 in chain C). The mesh is contoured at 1.6 in Coot, showing well-defined density for aromatic residues.]]
+| [[Image:022_Fig2.jpg|thumb|left|250px|Cryo-EM density map and model fitting of the hexameric ArnA complex. (a) ArnA hexamer reconstructed at 4.0 Å resolution, shown from three orientations: top, side, and bottom views. The map reveals the characteristic two-layered architecture of ArnA and clearly resolved secondary-structure elements. (b) Final atomic model of ArnA  fitted into the cryo-EM density map. Each of the 12 subunits is displayed in a different color to illustrate the hexameric arrangement. (c) Structural alignment of the final cryo-EM model (blue) (PDB ID [[9y5h]]) with the reference crystal structure (yellow) (PDB ID [[6pih]]) using ChimeraX. The alignment yielded an rmsd of 1.2 Å. Enlarged views show local conformational deviations in loop regions: Glu69–Ala98 in chain D (left) and Pro65–Ser75 in chain F (right). (d) Close-up view showing the model-to-map fit for a peptide segment (Leu483–Arg460). The density mesh is contoured at 1.7σ , showing clear peptide backbone density and supporting accurate model placement.]]   [[Image:022_Fig3.jpg|thumb|right|210px|Cryo-EM density map and model fitting of the AcrB trimer. (a) AcrB reconstructed at 2.92 Å resolution, shown from top, side, and bottom views. It reveals secondary-structure elements and transmembrane helices, consistent with the known trimeric architecture. (b) Final atomic model of AcrB fitted into the cryo-EM density map. Each protomer is shown in a different color to highlight the asymmetric trimer organization. (c) Structural alignment of the final cryo-EM model (blue)(PDB-ID [[9v5r]]) with the reference crystal structure , (yellow) (PDB ID [[7rr7]]), performed using ChimeraX. The rmsd between the structures is 1.2 Å. Enlarged views highlight conformational deviations, including E693–D711 in protomer B (left) and A704–D711 in protomer C (right), located within the PN2 subdomain. (d) Close-up view showing the density fit for side chains (W187, chain A, F572, chain B, W772. chain C). The mesh is contoured at 1.6σ in Coot, showing well-defined density for aromatic residues.]]
 |}

Journal:Acta Cryst D:S2059798325007089

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Revision as of 08:31, 18 September 2025

Off-Target Structural Insights: ArnA and AcrB in Bacterial Membrane Protein Cryo-EM Analysis

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