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- PDB-9rsj: Cryo-EM structure of MATE transporter NorM-VC in complex with dox... -

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Basic information

Entry
Database: PDB / ID: 9rsj
TitleCryo-EM structure of MATE transporter NorM-VC in complex with doxorubicin
Components
  • Anti-Fab nanobody
  • Multidrug resistance protein NorM
  • NabFab HC
  • NabFab LC
  • NorM-Nb17_4
KeywordsTRANSPORT PROTEIN / NabFab / substrate / multidrug transporter
Function / homology
Function and homology information


antiporter activity / xenobiotic transmembrane transporter activity / monoatomic ion transport / plasma membrane
Similarity search - Function
: / : / Multi antimicrobial extrusion protein / MatE
Similarity search - Domain/homology
DOXORUBICIN / Multidrug resistance protein NorM
Similarity search - Component
Biological speciesVibrio cholerae (bacteria)
synthetic construct (others)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsRomane, K. / Hsieh, P.Y. / Kowal, J. / Locher, K.P. / van Veen, H.W.
Funding support Switzerland, United Kingdom, 2items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_214834 Switzerland
Biotechnology and Biological Sciences Research Council (BBSRC)BB/K017713/1 United Kingdom
CitationJournal: J Mol Biol / Year: 2025
Title: Doxorubicin recognition and transport by the MATE multidrug transporter NorM from Vibrio cholerae.
Authors: Pei-Yu Hsieh / Ksenija Romane / Julia Kowal / Kaspar P Locher / Hendrik W van Veen /
Abstract: Multidrug and toxic compound extrusion (MATE) transport proteins contribute to multidrug resistance in human pathogens by extruding various cytotoxic compounds from the cellular interior. Despite ...Multidrug and toxic compound extrusion (MATE) transport proteins contribute to multidrug resistance in human pathogens by extruding various cytotoxic compounds from the cellular interior. Despite their importance across all domains of life, the specificities and mechanisms of substrate transport of these proteins remain poorly understood due to limited structural and functional information. Here, we determined the cryo-electron microscopy structure of NorM from Vibrio cholerae (NorM-VC) in complex with the anthracycline antibiotic doxorubicin, using the NabFab approach. The structure reveals that the doxorubicin-binding pocket is located halfway through the membrane, within the C-lobe of the protein. Functional studies targeting the doxorubicin-interacting residues validated the binding pocket and enabled detailed analysis of the doxorubicin transport reaction. Our findings indicate doxorubicin binding within a multisite binding chamber engaged in a general transport mechanism for a variety of substrates.
History
DepositionJul 1, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 26, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Multidrug resistance protein NorM
H: NabFab HC
L: NabFab LC
N: NorM-Nb17_4
K: Anti-Fab nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)127,1276
Polymers126,5835
Non-polymers5441
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Antibody , 4 types, 4 molecules HLNK

#2: Antibody NabFab HC


Mass: 25684.463 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#3: Antibody NabFab LC


Mass: 23258.783 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#4: Antibody NorM-Nb17_4


Mass: 14356.979 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Nanobody / Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#5: Antibody Anti-Fab nanobody


Mass: 13390.644 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Nanobody / Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)

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Protein / Non-polymers , 2 types, 2 molecules A

#1: Protein Multidrug resistance protein NorM / Multidrug-efflux transporter / Na(+)/drug antiporter


Mass: 49892.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Gene: VCM66_1481 / Production host: Escherichia coli (E. coli) / References: UniProt: C3LML9
#6: Chemical ChemComp-DM2 / DOXORUBICIN / ADRIAMYCIN


Mass: 543.519 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C27H29NO11 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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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: MATE transporter NorM from Vibrio cholerae in complex with NabFab, Nb17_4, anti-Fab nanobody and doxorubicin
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.128 MDa / Experimental value: NO
Source (natural)Organism: Vibrio cholerae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
7Cootmodel fitting
12cryoSPARC3D reconstruction
13PHENIX1.21_5207model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 327809 / Symmetry type: POINT
RefinementHighest resolution: 3.1 Å / Cross valid method: NONE
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038866
ELECTRON MICROSCOPYf_angle_d0.62412059
ELECTRON MICROSCOPYf_dihedral_angle_d11.2323112
ELECTRON MICROSCOPYf_chiral_restr0.0411381
ELECTRON MICROSCOPYf_plane_restr0.0061507

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