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- EMDB-64793: cryo-EM structure of trimeric AcrB -

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Basic information

Entry
Database: EMDB / ID: EMD-64793
Titlecryo-EM structure of trimeric AcrB
Map data
Sample
  • Complex: Multidrug efflux pump subunit AcrB
    • Protein or peptide: Multidrug efflux pump subunit AcrB
KeywordsMultidrug efflux pump / Trimeric transporter / RND transporter / Proton motive force / Active transport / Antimicrobial resistance / Drug efflux / Inner membrane transporter / AcrAB-TolC complex / TRANSPORT PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsCaliseki M / Borucu U / Kadapalakere SY / Schaffitzel C / Kabasakal BV
Funding support Turkey, United Kingdom, 5 items
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 SKN / Schaffitzel C / Kabasakal BV
History
DepositionMay 26, 2025-
Header (metadata) releaseJul 30, 2025-
Map releaseJul 30, 2025-
UpdateOct 8, 2025-
Current statusOct 8, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_64793.png

Downloads & links

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Map

FileDownload / File: emd_64793.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 360 pix.
= 378. Å		1.05 Å/pix.
x 360 pix.
= 378. Å		1.05 Å/pix.
x 360 pix.
= 378. Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.000147
Minimum - Maximum-0.0017514594 - 2.4508252
Average (Standard dev.)0.0012912292 (±0.027567418)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 377.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_64793_half_map_1.map
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Half map: #2

Fileemd_64793_half_map_2.map
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Sample components

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Entire : Multidrug efflux pump subunit AcrB

EntireName: Multidrug efflux pump subunit AcrB
Components
  • Complex: Multidrug efflux pump subunit AcrB
    • Protein or peptide: Multidrug efflux pump subunit AcrB

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Supramolecule #1: Multidrug efflux pump subunit AcrB

SupramoleculeName: Multidrug efflux pump subunit AcrB / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Multidrug efflux pump subunit AcrB

MacromoleculeName: Multidrug efflux pump subunit AcrB / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 111.823258 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPNFFIDRPI FAWVIAIIIM LAGGLAILKL PVAQYPTIAP PAVTISASYP GADAKTVQDT VTQVIEQNMN GIDNLMYMSS NSDSTGTVQ ITLTFESGTD ADIAQVQVQN KLQLAMPLLP QEVQQQGVSV EKSSSSFLMV VGVINTDGTM TQEDISDYVA A NMKDAISR ...String:
MPNFFIDRPI FAWVIAIIIM LAGGLAILKL PVAQYPTIAP PAVTISASYP GADAKTVQDT VTQVIEQNMN GIDNLMYMSS NSDSTGTVQ ITLTFESGTD ADIAQVQVQN KLQLAMPLLP QEVQQQGVSV EKSSSSFLMV VGVINTDGTM TQEDISDYVA A NMKDAISR TSGVGDVQLF GSQYAMRIWM NPNELNKFQL TPVDVITAIK AQNAQVAAGQ LGGTPPVKGQ QLNASIIAQT RL TSTEEFG KILLKVNQDG SRVLLRDVAK IELGGENYDI IAEFNGQPAS GLGIKLATGA NALDTAAAIR AELAKMEPFF PSG LKIVYP YDTTPFVKIS IHEVVKTLVE AIILVFLVMY LFLQNFRATL IPTIAVPVVL LGTFAVLAAF GFSINTLTMF GMVL AIGLL VDDAIVVVEN VERVMAEEGL PPKEATRKSM GQIQGALVGI AMVLSAVFVP MAFFGGSTGA IYRQFSITIV SAMAL SVLV ALILTPALCA TMLKPIAKGD HGEGKKGFFG WFNRMFEKST HHYTDSVGGI LRSTGRYLVL YLIIVVGMAY LFVRLP SSF LPDEDQGVFM TMVQLPAGAT QERTQKVLNE VTHYYLTKEK NNVESVFAVN GFGFAGRGQN TGIAFVSLKD WADRPGE EN KVEAITMRAT RAFSQIKDAM VFAFNLPAIV ELGTATGFDF ELIDQAGLGH EKLTQARNQL LAEAAKHPDM LTSVRPNG L EDTPQFKIDI DQEKAQALGV SINDINTTLG AAWGGSYVND FIDRGRVKKV YVMSEAKYRM LPDDIGDWYV RAADGQMVP FSAFSSSRWE YGSPRLERYN GLPSMEILGQ AAPGKSTGEA MELMEQLASK LPTGVGYDWT GMSYQERLSG NQAPSLYAIS LIVVFLCLA ALYESWSIPF SVMLVVPLGV IGALLAATFR GLTNDVYFQV GLLTTIGLSA KNAILIVEFA KDLMDKEGKG L IEATLDAV RMRLRPILMT SLAFILGVMP LVISTGAGSG AQNAVGTGVM GGMVTATVLA IFFVPVFFVV VRRRFS

UniProtKB: Multidrug efflux pump subunit AcrB

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 60.6 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7rr7

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 77800
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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