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- EMDB-10319: Cryo-EM structure of the full-length ABC-transporter IrtAB -

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Basic information

Entry
Database: EMDB / ID: EMD-10319
TitleCryo-EM structure of the full-length ABC-transporter IrtAB
Map dataPost-processed final 3D reconstruction of wildtype full-length IrtAB.
Sample
  • Complex: Full-length wildtype IrtAB complex
Biological speciesMycolicibacterium thermoresistibile (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.88 Å
AuthorsWeber MS / Arnold F / Gonda I / Seeger M / Medalia O
Funding support Switzerland, 3 items
OrganizationGrant numberCountry
Swiss National Science FoundationPP00P3_144823 Switzerland
Swiss National Science FoundationSNSF 31003A_179418 Switzerland
European Research Council772190 Switzerland
CitationJournal: 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
DepositionSep 19, 2019-
Header (metadata) releaseFeb 26, 2020-
Map releaseFeb 26, 2020-
UpdateFeb 10, 2021-
Current statusFeb 10, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0133
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0133
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10319.png

Downloads & links

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Map

FileDownload / File: emd_10319.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed final 3D reconstruction of wildtype full-length IrtAB.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 200 pix.
= 220. Å		1.1 Å/pix.
x 200 pix.
= 220. Å		1.1 Å/pix.
x 200 pix.
= 220. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0133 / Movie #1: 0.0133
Minimum - Maximum-0.03567526 - 0.05580336
Average (Standard dev.)-7.759846e-05 (±0.0025887615)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 220.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z220.000220.000220.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0360.056-0.000

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Supplemental data

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Half map: Unfiltered half map 1 of 3D Refinement

Fileemd_10319_half_map_1.map
AnnotationUnfiltered half map 1 of 3D Refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map 2 of 3D Refinement

Fileemd_10319_half_map_2.map
AnnotationUnfiltered half map 2 of 3D Refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Full-length wildtype IrtAB complex

EntireName: Full-length wildtype IrtAB complex
Components
  • Complex: Full-length wildtype IrtAB complex

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Supramolecule #1: Full-length wildtype IrtAB complex

SupramoleculeName: Full-length wildtype IrtAB complex / type: complex / ID: 1 / Parent: 0 / Details: Solubilized in beta-DDM
Source (natural)Organism: Mycolicibacterium thermoresistibile (bacteria) / Location in cell: inner plasma membrane
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: MC1061 / Recombinant plasmid: pBXC3GH
Molecular weightTheoretical: 158.736 KDa

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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

Concentration2.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMTris/HCLTris(hydroxymethyl)aminomethane hydrochloride
150.0 mMNaClsodium chloride
0.015 %beta-DDMn-Dodecyl beta-D-maltoside
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 6 s blotting time.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 45455
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

Particle selectionNumber selected: 335045
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: EMDB MAP
EMDB ID:

6007

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.88 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 70257
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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