+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-10319 | ||||||||||||
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Title | Cryo-EM structure of the full-length ABC-transporter IrtAB | ||||||||||||
Map data | Post-processed final 3D reconstruction of wildtype full-length IrtAB. | ||||||||||||
Sample |
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Biological species | Mycolicibacterium thermoresistibile (bacteria) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 6.88 Å | ||||||||||||
Authors | Weber MS / Arnold F / Gonda I / Seeger M / Medalia O | ||||||||||||
Funding support | Switzerland, 3 items
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Citation | Journal: Nature / Year: 2020 Title: The ABC exporter IrtAB imports and reduces mycobacterial siderophores. Authors: Fabian M Arnold / Miriam S Weber / Imre Gonda / Marc J Gallenito / Sophia Adenau / Pascal Egloff / Iwan Zimmermann / Cedric A J Hutter / Lea M Hürlimann / Eike E Peters / Jörn Piel / ...Authors: Fabian M Arnold / Miriam S Weber / Imre Gonda / Marc J Gallenito / Sophia Adenau / Pascal Egloff / Iwan Zimmermann / Cedric A J Hutter / Lea M Hürlimann / Eike E Peters / Jörn Piel / Gabriele Meloni / Ohad Medalia / Markus A Seeger / Abstract: Intracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores that scavenge iron from host proteins. M. ...Intracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores that scavenge iron from host proteins. M. tuberculosis produces two classes of siderophore, lipid-bound mycobactin and water-soluble carboxymycobactin. Functional studies have revealed that iron-loaded carboxymycobactin is imported into the cytoplasm by the ATP binding cassette (ABC) transporter IrtAB, which features an additional cytoplasmic siderophore interaction domain. However, the predicted ABC exporter fold of IrtAB is seemingly contradictory to its import function. Here we show that membrane-reconstituted IrtAB is sufficient to import mycobactins, which are then reduced by the siderophore interaction domain to facilitate iron release. Structure determination by X-ray crystallography and cryo-electron microscopy not only confirms that IrtAB has an ABC exporter fold, but also reveals structural peculiarities at the transmembrane region of IrtAB that result in a partially collapsed inward-facing substrate-binding cavity. The siderophore interaction domain is positioned in close proximity to the inner membrane leaflet, enabling the reduction of membrane-inserted mycobactin. Enzymatic ATPase activity and in vivo growth assays show that IrtAB has a preference for mycobactin over carboxymycobactin as its substrate. Our study provides insights into an unusual ABC exporter that evolved as highly specialized siderophore-import machinery in mycobacteria. | ||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_10319.map.gz | 28.2 MB | EMDB map data format | |
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Header (meta data) | emd-10319-v30.xml emd-10319.xml | 17.5 KB 17.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_10319_fsc.xml | 7.2 KB | Display | FSC data file |
Images | emd_10319.png | 82.6 KB | ||
Others | emd_10319_half_map_1.map.gz emd_10319_half_map_2.map.gz | 23.4 MB 23.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-10319 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-10319 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_10319.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Post-processed final 3D reconstruction of wildtype full-length IrtAB. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.1 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: Unfiltered half map 1 of 3D Refinement
File | emd_10319_half_map_1.map | ||||||||||||
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Annotation | Unfiltered half map 1 of 3D Refinement | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Unfiltered half map 2 of 3D Refinement
File | emd_10319_half_map_2.map | ||||||||||||
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Annotation | Unfiltered half map 2 of 3D Refinement | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Full-length wildtype IrtAB complex
Entire | Name: Full-length wildtype IrtAB complex |
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Components |
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-Supramolecule #1: Full-length wildtype IrtAB complex
Supramolecule | Name: Full-length wildtype IrtAB complex / type: complex / ID: 1 / Parent: 0 / Details: Solubilized in beta-DDM |
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Source (natural) | Organism: Mycolicibacterium thermoresistibile (bacteria) / Location in cell: inner plasma membrane |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: MC1061 / Recombinant plasmid: pBXC3GH |
Molecular weight | Theoretical: 158.736 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 2.5 mg/mL | ||||||||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: AIR | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 6 s blotting time. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 45455 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-50 / Number real images: 2507 / Average exposure time: 10.0 sec. / Average electron dose: 85.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |