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- EMDB-56885: NorA bound to miniprotein I-23 -

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Basic information

Entry
Database: EMDB / ID: EMD-56885
TitleNorA bound to miniprotein I-23
Map data
Sample
  • Complex: NorA bound to miniprotein I-23
    • Protein or peptide: I23 miniprotein
    • Protein or peptide: Quinolone resistance protein NorA,Soluble cytochrome b562
KeywordsMultidrug efflux transporter / efflux pump inhibitor / TRANSPORT PROTEIN
Function / homology
Function and homology information


xenobiotic transmembrane transporter activity / electron transport chain / periplasmic space / electron transfer activity / iron ion binding / heme binding / plasma membrane
Similarity search - Function
: / Tetracycline resistance protein TetA/multidrug resistance protein MdtG / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / MFS transporter superfamily
Similarity search - Domain/homology
Soluble cytochrome b562 / Quinolone resistance protein NorA
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria) / Synthetic construct (others) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.79 Å
AuthorsLamon G / Mishra P / Chazin-Gray A / Baker D / Traaseth NJ
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI165782 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI108889 United States
CitationJournal: bioRxiv / Year: 2026
Title: Miniprotein inhibitors of the efflux transporter NorA.
Authors: Priyanka Mishra / Adam Chazin-Gray / Gaëlle Lamon / David Kim / David Baker / Nathaniel J Traaseth /
Abstract: Multidrug efflux pumps transport antibiotics across the cellular membrane resulting in resistance conferred to the host organism. Efflux pump inhibitors (EPIs) potentiate the efficacy of antibiotics ...Multidrug efflux pumps transport antibiotics across the cellular membrane resulting in resistance conferred to the host organism. Efflux pump inhibitors (EPIs) potentiate the efficacy of antibiotics by blocking drug efflux and hold promise as adjuvant therapeutics in the fight against multidrug resistant pathogenic bacteria. A hurdle in the field has been the lack of selectivity of small molecule EPIs which often display off-target toxicity due to non-specific binding. To tackle this specificity challenge, we aimed to maximize an inhibitor's binding surface area to efflux pumps by designing miniprotein EPIs using computational protein design and an co-expression assay to screen inhibition in cells. We used NorA as a model efflux transporter since it confers drug resistance to fluoroquinolones, puromycin, and other cytotoxic compounds. Starting from a focused miniprotein library of only 86 members, we identified inhibitors in the screen that blocked NorA transport under active efflux conditions . Our most promising inhibitor I-23 was validated by solving a cryo-EM structure of the miniprotein in complex with NorA, which stabilized the transporter in the outward-open conformation. I-23 has a ferredoxin-like fold with one of its β-hairpins inserted into the substrate binding pocket of NorA and other parts of the globular fold occupying the shallow pocket and making extensive intermolecular contacts with NorA. An arginine residue on the tip of the hairpin loop was positioned near an anionic patch required for NorA antibiotic efflux. The identified structural motifs in this work could be employed to explore the molecular properties of peptidoglycan penetration; full realization of the therapeutic potential of the designed miniprotein inhibitors will require determining the principles for facilitating passage of ~7 to 8 kDa miniproteins across the peptidoglycan bacterial cell wall.
History
DepositionFeb 21, 2026-
Header (metadata) releaseApr 8, 2026-
Map releaseApr 8, 2026-
UpdateApr 8, 2026-
Current statusApr 8, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_56885.png

Downloads & links

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Map

FileDownload / File: emd_56885.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 300 pix.
= 247.5 Å		0.83 Å/pix.
x 300 pix.
= 247.5 Å		0.83 Å/pix.
x 300 pix.
= 247.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.756
Minimum - Maximum-2.7000804 - 4.463601
Average (Standard dev.)0.0007672608 (±0.070579916)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 247.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_56885_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_56885_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NorA bound to miniprotein I-23

EntireName: NorA bound to miniprotein I-23
Components
  • Complex: NorA bound to miniprotein I-23
    • Protein or peptide: I23 miniprotein
    • Protein or peptide: Quinolone resistance protein NorA,Soluble cytochrome b562

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Supramolecule #1: NorA bound to miniprotein I-23

SupramoleculeName: NorA bound to miniprotein I-23 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Staphylococcus aureus (bacteria)

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Macromolecule #1: I23 miniprotein

MacromoleculeName: I23 miniprotein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synthetic construct (others)
Molecular weightTheoretical: 7.999065 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GGMYEVIVRN APRSFVKEVR EETGAKVSRT YINLNRISAV FTTFTHERKE DAEAIAERAR ARGLEVFLVE

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Macromolecule #2: Quinolone resistance protein NorA,Soluble cytochrome b562

MacromoleculeName: Quinolone resistance protein NorA,Soluble cytochrome b562
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 53.388516 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNKQIFVLYF NIFLIFLGIG LVIPVLPVYL KDLGLTGSDL GLLVAAFALS QMIISPFGGT LADKLGKKLI ICIGLILFSV SEFMFAVGH NFSVLMLSRV IGGMSAGMVM PGVTGLIADI SPSHQKAKNF GYMSAIINSG FILGPGIGGF MAEVSHRMPF Y FAGALGIL ...String:
MNKQIFVLYF NIFLIFLGIG LVIPVLPVYL KDLGLTGSDL GLLVAAFALS QMIISPFGGT LADKLGKKLI ICIGLILFSV SEFMFAVGH NFSVLMLSRV IGGMSAGMVM PGVTGLIADI SPSHQKAKNF GYMSAIINSG FILGPGIGGF MAEVSHRMPF Y FAGALGIL AFIMSIVLIH DPKKSTTSGF QKLEPQLLTK INWKVFITPV ILTLVLSFGL SAFETLYSLY TADKVNYSPK DI SIAITGG GIFGALFQIY FFDKFMKYFS ELTFIAWSLL YSVVVLILLV FANDYWSIML ISFVVFIGFD MIRPAITNYF SNI AGERQG FAGGLNSTFT SMGNFIGPLI AGALFDVHIE APIYMAIGVS LAGVVIVLIE KQHRAAAADL EDNWETLNDN LKVI EKADN AAQVKDALTK MRAAALDAQK ATPPKLEDKS PDSPEMKDFR HGFDILVGQI DDALKLANEG KVKEAQAAAE QLKTT RNAY IQKYL

UniProtKB: Quinolone resistance protein NorA, Soluble cytochrome b562

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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

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 49.59 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 302069
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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