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- PDB-9g2s: Cryo-EM structure of IrtAB 2xEQ, Q249R_IrtB mutant in LMNG -

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Basic information

Entry
Database: PDB / ID: 9g2s
TitleCryo-EM structure of IrtAB 2xEQ, Q249R_IrtB mutant in LMNG
Components
  • Mycobactin import ATP-binding/permease protein IrtA
  • Mycobactin import ATP-binding/permease protein IrtB
KeywordsMEMBRANE PROTEIN / ABC transporter / type IV ABC importer siderophore / mycobactin / heterodimeric ABC transporter
Function / homology
Function and homology information


Translocases; Catalysing the translocation of inorganic cations; Linked to the hydrolysis of a nucleoside triphosphate / ATPase-coupled lipid transmembrane transporter activity / ABC-type transporter activity / oxidoreductase activity / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
Siderophore-interacting protein, C-terminal domain / FAD-binding 9, siderophore-interacting / Siderophore-interacting protein / Siderophore-interacting FAD-binding domain / Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / FAD-binding domain, ferredoxin reductase-type ...Siderophore-interacting protein, C-terminal domain / FAD-binding 9, siderophore-interacting / Siderophore-interacting protein / Siderophore-interacting FAD-binding domain / Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / Riboflavin synthase-like beta-barrel / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Mycobactin import ATP-binding/permease protein IrtA / Mycobactin import ATP-binding/permease protein IrtB
Similarity search - Component
Biological speciesMycolicibacterium thermoresistibile ATCC 19527 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsGonda, I. / Seeger, M.A.
Funding support Switzerland, European Union, 4items
OrganizationGrant numberCountry
University of ZurichFK-19-028 Switzerland
European Research Council (ERC)consolidator grant 772190European Union
Swiss National Science FoundationPP00P3_144823 Switzerland
Swiss National Science Foundation310030_188817 Switzerland
CitationJournal: Nat Commun / Year: 2025
Title: The mycobacterial ABC transporter IrtAB employs a membrane-facing crevice for siderophore-mediated iron uptake.
Authors: Imre Gonda / Simona Sorrentino / Laura Galazzo / Nicolas P Lichti / Fabian M Arnold / Ahmad R Mehdipour / Enrica Bordignon / Markus A Seeger /
Abstract: The mycobacterial ABC transporter IrtAB features an ABC exporter fold, yet it imports iron-charged siderophores called mycobactins. Here, we present extensive cryo-EM analyses and DEER measurements, ...The mycobacterial ABC transporter IrtAB features an ABC exporter fold, yet it imports iron-charged siderophores called mycobactins. Here, we present extensive cryo-EM analyses and DEER measurements, revealing that IrtAB alternates between an inward-facing and an outward-occluded conformation, but does not sample an outward-facing conformation. When IrtAB is locked in its outward-occluded conformation in nanodiscs, mycobactin is bound in the middle of the lipid bilayer at a membrane-facing crevice opening at the heterodimeric interface. Mutations introduced at the crevice abrogate mycobactin import and in corresponding structures, the crevice is collapsed. A conserved triple histidine motif coordinating a zinc ion is present below the mycobactin binding site. Substitution of these histidine residues with alanine results in a decoupled transporter, which hydrolyzes ATP, but lost its capacity to import mycobactins. Our data suggest that IrtAB imports mycobactin via a credit-card mechanism in a transport cycle that is coupled to the presence of zinc.
History
DepositionJul 11, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 18, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 12, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_abbrev / _citation.journal_id_ASTM ..._citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Mycobactin import ATP-binding/permease protein IrtA
B: Mycobactin import ATP-binding/permease protein IrtB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)127,0256
Polymers125,9622
Non-polymers1,0634
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Mycobactin import ATP-binding/permease protein IrtA


Mass: 64374.340 Da / Num. of mol.: 1 / Mutation: E815Q
Source method: isolated from a genetically manipulated source
Details: SID domain is truncated (IrtA 1-314)
Source: (gene. exp.) Mycolicibacterium thermoresistibile ATCC 19527 (bacteria)
Gene: irtA, KEK_01485 / Production host: Escherichia coli MC1061 (bacteria)
References: UniProt: G7CBF5, Translocases; Catalysing the translocation of inorganic cations; Linked to the hydrolysis of a nucleoside triphosphate
#2: Protein Mycobactin import ATP-binding/permease protein IrtB


Mass: 61587.617 Da / Num. of mol.: 1 / Mutation: E493Q, Q249R
Source method: isolated from a genetically manipulated source
Details: contains cleaved, C-terminal 3C enzyme recognition site
Source: (gene. exp.) Mycolicibacterium thermoresistibile ATCC 19527 (bacteria)
Gene: irtB, KEK_01490 / Production host: Escherichia coli MC1061 (bacteria)
References: UniProt: G7CBF6, Translocases; Catalysing the translocation of inorganic cations; Linked to the hydrolysis of a nucleoside triphosphate
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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: IrtAB / Type: COMPLEX
Details: IrtAB 2xEQ mutant with IrtB-Q249R mutation, devoid the SID, in the presence of ATP-Mg, mycobactin, solubilized in LMNG detergent
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.126 MDa / Experimental value: NO
Source (natural)Organism: Mycolicibacterium thermoresistibile ATCC 19527 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: MC1061
Buffer solutionpH: 7.5
Details: 20mM Tris-HCl pH 7.5, 150mM NaCl, 0.01% LMNG, 20uM mycobactin
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMTRISC4H11NO31
32.5 mMATPC10H16N5O13P31
42.5 mMmagnesium chlorideMgCl21
50.01 w/v%LMNGC47H88O221
SpecimenConc.: 3.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 190000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 20 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.13 sec. / Electron dose: 52.5 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7400
Details: micrographs were collected with 47 frames per movie
Image scansWidth: 4096 / Height: 4096

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1779249
Details: bona fide particles following on-the-fly 2D classification
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 151484 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 6TEJ

6tej


Accession code: 6TEJ / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 44.86 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00358663
ELECTRON MICROSCOPYf_angle_d0.623111832
ELECTRON MICROSCOPYf_chiral_restr0.0441441
ELECTRON MICROSCOPYf_plane_restr0.00461510
ELECTRON MICROSCOPYf_dihedral_angle_d12.13543162

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