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- PDB-9nn6: E. coli Cir in Complex with the RBD of Microcin V -

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Basic information

Entry
Database: PDB / ID: 9nn6
TitleE. coli Cir in Complex with the RBD of Microcin V
Components
  • Colicin I receptor
  • Colicin-V
KeywordsMEMBRANE PROTEIN / TonB-dependent receptor / beta barrel / antimicrobial protein / MccV
Function / homology
Function and homology information


siderophore-iron transmembrane transporter activity / colicin transmembrane transporter activity / siderophore transmembrane transport / siderophore uptake transmembrane transporter activity / transmembrane transporter complex / cell outer membrane / killing of cells of another organism / intracellular iron ion homeostasis / defense response to bacterium / extracellular region / membrane
Similarity search - Function
Microcin V bacteriocin / Microcin V bacteriocin / TonB-dependent receptor (TBDR) proteins signature 1. / TonB box, conserved site / TonB-dependent receptor, conserved site / TonB-dependent receptor (TBDR) proteins signature 2. / TonB-dependent receptor-like, beta-barrel / TonB dependent receptor-like, beta-barrel / TonB-dependent receptor (TBDR) proteins profile. / Vitamin B12 transporter BtuB-like ...Microcin V bacteriocin / Microcin V bacteriocin / TonB-dependent receptor (TBDR) proteins signature 1. / TonB box, conserved site / TonB-dependent receptor, conserved site / TonB-dependent receptor (TBDR) proteins signature 2. / TonB-dependent receptor-like, beta-barrel / TonB dependent receptor-like, beta-barrel / TonB-dependent receptor (TBDR) proteins profile. / Vitamin B12 transporter BtuB-like / TonB-dependent receptor, plug domain superfamily / TonB-dependent receptor, plug domain / TonB-dependent Receptor Plug Domain / TonB-dependent receptor-like, beta-barrel domain superfamily
Similarity search - Domain/homology
Colicin I receptor / Colicin-V
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Enterobacteriaceae (enterobacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsMaurakis, S.A. / Botos, I. / Buchanan, S.K.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK) United States
CitationJournal: Commun Biol / Year: 2025
Title: Structural insights into Cir-mediated killing by the antimicrobial protein Microcin V.
Authors: Stavros A Maurakis / Angela C O'Donnell / Istvan Botos / Rodolfo Ghirlando / Bryan W Davies / Susan K Buchanan /
Abstract: Drug-resistant bacteria are a global concern. Novel treatments are needed, but are difficult to develop for Gram-negative species due to the need to traverse the outer membrane to reach targets ...Drug-resistant bacteria are a global concern. Novel treatments are needed, but are difficult to develop for Gram-negative species due to the need to traverse the outer membrane to reach targets beneath. A promising solution is found in natural antibiotics which bind outer membrane receptors and co-opt them for import. Exploring this mechanism may open avenues for antibiotic development. An underappreciated class of natural antibiotics are microcins - small antimicrobial proteins secreted by certain bacteria during inter-species competition. Microcins bind outer-membrane receptors of prey species for passage into the periplasm. They have potent activity, bind specific targets, and can control pathobiont expansion and colonization. One microcin, MccV, utilizes the E. coli colicin Ia receptor, Cir, for import. Here, we report the first high-resolution structure of the Cir/MccV complex by Cryo-EM, revealing an interaction centered on an electropositive cavity within the Cir extracellular loops. We also report the affinity of MccV for Cir. Lastly, we mutagenized interacting residues and identified key contacts critical to MccV binding, import, and bacteriolysis. Future efforts may help disentangle the mechanisms of microcin killing and will assess relationships between other microcins and their targets to better understand the potential for microcins to be used as antibacterial drugs.
History
DepositionMar 5, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 24, 2025Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Oct 22, 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 / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Colicin I receptor
B: Colicin-V


Theoretical massNumber of molelcules
Total (without water)74,1422
Polymers74,1422
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 Colicin I receptor


Mass: 70576.594 Da / Num. of mol.: 1 / Mutation: W307M, L312M, F558M, V560M
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: cirA, cir, feuA, b2155, JW2142 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P17315
#2: Protein/peptide Colicin-V / Microcin-V bacteriocin


Mass: 3564.979 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteriaceae (enterobacteria) / Gene: cvaC / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P22522
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: E. coli Cir in Complex with the RBD of Microcin V / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3)
Buffer solutionpH: 7.4 / Details: Phosphate Buffered Saline
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Blot force +5 Wait time 0 Blot total 1 Blot time 5 seconds

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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: 800 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 59.65 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
2SerialEMimage acquisition
4cryoSPARC4CTF correction
9cryoSPARC4initial Euler assignment
10cryoSPARC4final Euler assignment
11cryoSPARC4classification
12cryoSPARC43D reconstruction
13PHENIX1.20.1_4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 102419 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 2.9 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0025334
ELECTRON MICROSCOPYf_angle_d0.3817245
ELECTRON MICROSCOPYf_dihedral_angle_d3.264730
ELECTRON MICROSCOPYf_chiral_restr0.041773
ELECTRON MICROSCOPYf_plane_restr0.003957

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