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- EMDB-50978: Cryo-EM structure of IrtAB in inward-facing state in LMNG -

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Basic information

Entry
Database: EMDB / ID: EMD-50978
TitleCryo-EM structure of IrtAB in inward-facing state in LMNG
Map data
Sample
  • Complex: IrtAB
    • Protein or peptide: Mycobactin import ATP-binding/permease protein IrtA
    • Protein or peptide: Mycobactin import ATP-binding/permease protein IrtB
  • Ligand: ZINC ION
KeywordsABC transporter / type IV ABC importer siderophore / mycobactin / heterodimeric ABC transporter / MEMBRANE PROTEIN
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
Mycobactin import ATP-binding/permease protein IrtA / Mycobactin import ATP-binding/permease protein IrtB
Similarity search - Component
Biological speciesMycolicibacterium thermoresistibile ATCC 19527 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsGonda I / Seeger MA
Funding support Switzerland, European Union, 4 items
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, 2024-
Header (metadata) releaseDec 18, 2024-
Map releaseDec 18, 2024-
UpdateFeb 12, 2025-
Current statusFeb 12, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50978.png

Downloads & links

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Map

FileDownload / File: emd_50978.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 200 pix.
= 260. Å		1.3 Å/pix.
x 200 pix.
= 260. Å		1.3 Å/pix.
x 200 pix.
= 260. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.818
Minimum - Maximum-3.747331 - 5.869928
Average (Standard dev.)0.0012263077 (±0.12558006)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 260.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50978_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_50978_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_50978_half_map_2.map
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Sample components

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Entire : IrtAB

EntireName: IrtAB
Components
  • Complex: IrtAB
    • Protein or peptide: Mycobactin import ATP-binding/permease protein IrtA
    • Protein or peptide: Mycobactin import ATP-binding/permease protein IrtB
  • Ligand: ZINC ION

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Supramolecule #1: IrtAB

SupramoleculeName: IrtAB / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: wild-type, apo IrtAB in nanodisc
Source (natural)Organism: Mycolicibacterium thermoresistibile ATCC 19527 (bacteria)
Molecular weightTheoretical: 158.9 KDa

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

MacromoleculeName: Mycobactin import ATP-binding/permease protein IrtA / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Translocases; Catalysing the translocation of inorganic cations; Linked to the hydrolysis of a nucleoside triphosphate
Source (natural)Organism: Mycolicibacterium thermoresistibile ATCC 19527 (bacteria)
Molecular weightTheoretical: 97.365344 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: SLRGLGARDH QATVVDKEYI APHFVRVRLV SPTLFDEVIV EPTSWLRFWF PDPDGSDTEF QRAYTITESD PETGRFAVDM VLHEPAGPA STWARTVEPG ATIAVMSMGS RGFSVPEDPE DRPVGYLLIG DSASTPAING IIEVVPHDIP IELYLEQHHD D DVLIPLAE ...String:
SLRGLGARDH QATVVDKEYI APHFVRVRLV SPTLFDEVIV EPTSWLRFWF PDPDGSDTEF QRAYTITESD PETGRFAVDM VLHEPAGPA STWARTVEPG ATIAVMSMGS RGFSVPEDPE DRPVGYLLIG DSASTPAING IIEVVPHDIP IELYLEQHHD D DVLIPLAE HPRLRVHRVS RDDASSLAAA LELRDWSNWY CWAGPEAGAL KQVRTRLRDE FGFPKREVYA QAYWTEGRAM GS SRGETST PAKPAAKTAP AKAAAKPAAA SGAGTPEHAA APAAATTGAP QAAPAPGAAQ PRTPVRGRWR AEAGSRLLAP LKK PLIVSG VLQALITLIE LAPFVLLVEL ARLLLGGAEA ERLWTLGLTA VSLIGLGAVL AAAMTLWLHR VDARFAHELR GRLL TKLSR LPLGWFTRRG SASTKQLVQD DTLALHYLIT HAIPDAVAAV VAPVAVLVYL FVADWRVALV LFIPVLVYLV LMSVM TIQS GSKIAQAPRW AERMGGEAGA FLEGQPVIRI FGGAAASRFR RRLDDYIDFL VSWQRPFVGK KTLMDLVTRP ATFLWI ILV AGVPLVVTGR MDPVNLLPFL LLGTTFGARL LGIGYGLSGI QTGMLAARRI QTVLDEPELV VRDRTGQAGT DHASGDQ AR PGTVELDRVS FEYRPGVPVI RDVTLTLRPG TVTALVGPSG SGKSTLAALV ARFHDVTQGA IRVDGRDIRT LTADELYR R VGFVLQDAQL VHGSVAENIA LAEPDAGLER IRTAARDAQI HDRITRMPDG YDSVLGAGSA LSGGERQRVT IARAILADT PVLVLDEATA FADPESEYLV QQAINRLTRD RTVLVIAHRL HTITHADQIV VLDDGRIVEV GTHDELLAAG GRYRGLWDSG RYSSPDAGR PVSADAVEVG R

UniProtKB: Mycobactin import ATP-binding/permease protein IrtA

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Macromolecule #2: Mycobactin import ATP-binding/permease protein IrtB

MacromoleculeName: Mycobactin import ATP-binding/permease protein IrtB / type: protein_or_peptide / ID: 2
Details: contains cleaved, C-terminal 3C enzyme recognition site
Number of copies: 1 / Enantiomer: LEVO
EC number: Translocases; Catalysing the translocation of inorganic cations; Linked to the hydrolysis of a nucleoside triphosphate
Source (natural)Organism: Mycolicibacterium thermoresistibile ATCC 19527 (bacteria)
Molecular weightTheoretical: 61.559539 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString: MIRTLLRLVP AEKRGAVAGY AVLTLLSVLL RAVGAVLLIP LLAALFSDTP SDAWLWLGWL TAVTLAGWVT DTNTARLGFD LGFAVLSRT QHDMADRLPN VAMSWFTPDN TATARQAIAA TGPELAGLVV NLLTPLIGAA LLPAAIGVAL LFVSVPLGLA A LAGVAVLF ...String:
MIRTLLRLVP AEKRGAVAGY AVLTLLSVLL RAVGAVLLIP LLAALFSDTP SDAWLWLGWL TAVTLAGWVT DTNTARLGFD LGFAVLSRT QHDMADRLPN VAMSWFTPDN TATARQAIAA TGPELAGLVV NLLTPLIGAA LLPAAIGVAL LFVSVPLGLA A LAGVAVLF GALALSGRLS RAADKVAGET NSAFTERIIE FARTQQALRA ARRVEPARSQ VGSALAAQHG AGLRLLTMQI PG QVLFSLA GQVALIGFAG MAVWLTVRGQ LGVPEAIALI VVLVRYLEPF AAIADLAPAL ETTRATLNRI QAVLDAPTLP AGR RRLDRT GAAPSIEFDD VRFSYGDEVV LDGVSFTLRP GNTTAIVGPS GSGKTTILSL IAGLQQPASG RVLLDGVDVT TLDP EARRA AVSVVFQHPY LFDGTLRDNV LVGDPEADPD DVTAAMRLAR VDELLDRLPD GDATVVGEGG TALSGGERQR VSIAR ALLK PAPVLLVDEA TSALDNANEA AVVDALTADP RPRTRVIVAH RLASIRHADR VLFVEAGRVV EDGAIDELLA AGGRFA QFW AQQQAASEWA IGSTARALEV LFQ

UniProtKB: Mycobactin import ATP-binding/permease protein IrtB

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration3.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMC4H11NO3TRIS
0.01 w/v%C47H88O22LMNG

Details: 20mM Tris-HCl pH 7.5, 150mM NaCl, 0.01% LMNG
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 283 K / Instrument: LEICA EM GP
DetailsSample was plunge-frozen without prior pretreatment.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 1 / Number real images: 7906 / Average exposure time: 0.93 sec. / Average electron dose: 59.9 e/Å2
Details: micrographs were collected in super-resolution mode, 38 frames per movie
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6tej

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 155919
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6tej


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL
Output model

PDB-9g2l:
Cryo-EM structure of IrtAB in inward-facing state in LMNG

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