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- PDB-9v5r: cryo-EM structure of trimeric AcrB -

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Basic information

Entry
Database: PDB / ID: 9v5r
Titlecryo-EM structure of trimeric AcrB
ComponentsMultidrug efflux pump subunit AcrB
KeywordsTRANSPORT PROTEIN / Multidrug efflux pump / Trimeric transporter / RND transporter / Proton motive force / Active transport / Antimicrobial resistance / Drug efflux / Inner membrane transporter / AcrAB-TolC complex
Function / homology
Function and homology information


alkane transmembrane transporter activity / alkane transport / enterobactin transport / enterobactin transmembrane transporter activity / xenobiotic detoxification by transmembrane export across the cell outer membrane / periplasmic side of plasma membrane / efflux pump complex / bile acid transmembrane transporter activity / xenobiotic transport / bile acid and bile salt transport ...alkane transmembrane transporter activity / alkane transport / enterobactin transport / enterobactin transmembrane transporter activity / xenobiotic detoxification by transmembrane export across the cell outer membrane / periplasmic side of plasma membrane / efflux pump complex / bile acid transmembrane transporter activity / xenobiotic transport / bile acid and bile salt transport / efflux transmembrane transporter activity / xenobiotic transmembrane transporter activity / fatty acid transport / response to toxic substance / response to xenobiotic stimulus / response to antibiotic / identical protein binding / membrane / plasma membrane
Similarity search - Function
Hydrophobe/amphiphile efflux-1 HAE1 / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / Acriflavin resistance protein / AcrB/AcrD/AcrF family
Similarity search - Domain/homology
Multidrug efflux pump subunit AcrB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsCaliseki, M. / Borucu, U. / Kadapalakere, S.Y. / Schaffitzel, C. / Kabasakal, B.V.
Funding support Turkey, United Kingdom, 5items
OrganizationGrant numberCountry
Other government118C225 Turkey
Biotechnology and Biological Sciences Research Council (BBSRC)BB/P000940/1 United Kingdom
Other government210701/Z/18/Z United Kingdom
Other government2211-A Turkey
Other government2214-A Turkey
Citation
Journal: Acta Crystallogr D Struct Biol / Year: 2025
Title: Off-target structural insights: ArnA and AcrB in bacterial membrane-protein cryo-EM analysis.
Authors: Mehmet Caliseki / Ufuk Borucu / Sathish K N Yadav / Christiane Schaffitzel / Burak Veli Kabasakal /
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.
#1: Journal: Biorxiv / Year: 2025
Title: Off-Target Structural Insights: ArnA and AcrB in Bacterial Membrane Protein Cryo-EM Analysis
Authors: Caliseki, M. / Borucu, U. / Yadav, S.K.N. / Schaffitzel, C. / Kabasakal, B.V.
History
DepositionMay 26, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 30, 2025Provider: repository / Type: Initial release
Revision 1.1Sep 24, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update
Revision 1.2Oct 8, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Multidrug efflux pump subunit AcrB
B: Multidrug efflux pump subunit AcrB
C: Multidrug efflux pump subunit AcrB


Theoretical massNumber of molelcules
Total (without water)335,4703
Polymers335,4703
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Multidrug efflux pump subunit AcrB / AcrAB-TolC multidrug efflux pump subunit AcrB / Acridine resistance protein B


Mass: 111823.258 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: acrB, acrE, b0462, JW0451 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P31224
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Multidrug efflux pump subunit AcrB / Type: COMPLEX / Entity ID: all / Source: NATURAL
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 60.6 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.2_5419: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77800 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00323584
ELECTRON MICROSCOPYf_angle_d0.79932049
ELECTRON MICROSCOPYf_dihedral_angle_d4.1433244
ELECTRON MICROSCOPYf_chiral_restr0.0443809
ELECTRON MICROSCOPYf_plane_restr0.0044093

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