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- EMDB-46916: Cryo-EM structure of the heme/hemoglobin transporter ChuA, in com... -

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Basic information

Entry
Database: EMDB / ID: EMD-46916
TitleCryo-EM structure of the heme/hemoglobin transporter ChuA, in complex with de novo designed binder G7
Map dataMain Sharpened Map
Sample
  • Complex: Complex between the heme/hemoglobin transporter ChuA and de novo designed binding protein G7
    • Protein or peptide: ChuA binding protein G7
    • Protein or peptide: Outer membrane heme/hemoglobin receptor
KeywordsTonB-dependent transporter / ChuA / Heme / Hemoglobin / Outer-membrane / de novo designed protein / binding protein / TRANSPORT PROTEIN
Function / homology
Function and homology information


heme transmembrane transporter activity / siderophore uptake transmembrane transporter activity / cell outer membrane
Similarity search - Function
TonB-dependent haem/haemoglobin receptor / TonB-dependent haemoglobin/transferrin/lactoferrin receptor / 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
Outer membrane heme/hemoglobin receptor
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / synthetic construct (others) / Escherichia coli CFT073 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsFox D / Venugopal H / Lupton CJ / Spicer BA / Grinter R
Funding support Australia, 2 items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)APP1197376 Australia
Australian Research Council (ARC)LE200100045, LE120100090 Australia
CitationJournal: Nat Commun / Year: 2025
Title: Inhibiting heme piracy by pathogenic Escherichia coli using de novo-designed proteins.
Authors: Daniel R Fox / Kazem Asadollahi / Imogen Samuels / Bradley A Spicer / Ashleigh Kropp / Christopher J Lupton / Kevin Lim / Chunxiao Wang / Hari Venugopal / Marija Dramicanin / Gavin J Knott / Rhys Grinter /
Abstract: Iron is an essential nutrient for most bacteria and is often growth-limiting during infection, due to the host sequestering free iron as part of the innate immune response. To obtain the iron ...Iron is an essential nutrient for most bacteria and is often growth-limiting during infection, due to the host sequestering free iron as part of the innate immune response. To obtain the iron required for growth, many bacterial pathogens encode transporters capable of extracting the iron-containing cofactor heme directly from host proteins. Pathogenic E. coli and Shigella spp. produce the outer membrane transporter ChuA, which binds host hemoglobin and extracts its heme cofactor, before importing heme into the cell. Heme extraction by ChuA is a dynamic process, with the transporter capable of rapidly extracting heme from hemoglobin in the absence of an external energy source, without forming a stable ChuA-hemoglobin complex. In this work, we utilise a combination of structural modelling, Cryo-EM, X-ray crystallography, mutagenesis, and phenotypic analysis to understand the mechanistic detail of this process. Based on this understanding we utilise artificial intelligence-based protein design to create binders capable of inhibiting E. coli growth by blocking hemoglobin binding to ChuA. By screening a limited number of these designs, we identify several binders that inhibit E. coli growth at low nanomolar concentrations, without experimental optimisation. We determine the structure of a subset of these binders, alone and in complex with ChuA, demonstrating that they closely match the computational design. This work demonstrates the utility of de novo-designed proteins for inhibiting bacterial nutrient uptake and uses a workflow that could be applied to integral membrane proteins in other organisms.
History
DepositionSep 5, 2024-
Header (metadata) releaseMay 21, 2025-
Map releaseMay 21, 2025-
UpdateJul 23, 2025-
Current statusJul 23, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46916.png

Downloads & links

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Map

FileDownload / File: emd_46916.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain Sharpened Map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.19 Å/pix.
x 220 pix.
= 261.8 Å		1.19 Å/pix.
x 220 pix.
= 261.8 Å		1.19 Å/pix.
x 220 pix.
= 261.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.19 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.257
Minimum - Maximum-2.5423913 - 3.8801413
Average (Standard dev.)0.000034137312 (±0.071547255)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 261.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_46916_additional_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_46916_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_46916_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Complex between the heme/hemoglobin transporter ChuA and de novo ...

EntireName: Complex between the heme/hemoglobin transporter ChuA and de novo designed binding protein G7
Components
  • Complex: Complex between the heme/hemoglobin transporter ChuA and de novo designed binding protein G7
    • Protein or peptide: ChuA binding protein G7
    • Protein or peptide: Outer membrane heme/hemoglobin receptor

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Supramolecule #1: Complex between the heme/hemoglobin transporter ChuA and de novo ...

SupramoleculeName: Complex between the heme/hemoglobin transporter ChuA and de novo designed binding protein G7
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli) / Strain: CFT073

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Macromolecule #1: ChuA binding protein G7

MacromoleculeName: ChuA binding protein G7 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.43385 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SIREKALKRN KEVLKLAKEI EKRTREALEE AKKIAEEGGE EGKKKAEEII KKTAKEVSEK VVEALRKGAE LAEAENPYAA KAAKKMRAN AEALEKLLKE DPRKALEEIL EMSEEAVKET EKKIKEMGLE HHHHHH

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Macromolecule #2: Outer membrane heme/hemoglobin receptor

MacromoleculeName: Outer membrane heme/hemoglobin receptor / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli CFT073 (bacteria)
Molecular weightTheoretical: 69.586219 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: TETMTVTATG NARSSFEAPM MVSVIDTSAP ENQTATSATD LLRHVPGITL DGTGRTNGQD VNMRGYDHRG VLVLVDGIRQ GTDTGHLNG TFLDPALIKR VEIVRGPSAL LYGSGALGGV ISYDTVDAKD LLQEGQSSGF RVFGTGGTGD HSLGLGASAF G RTENLDGI ...String:
TETMTVTATG NARSSFEAPM MVSVIDTSAP ENQTATSATD LLRHVPGITL DGTGRTNGQD VNMRGYDHRG VLVLVDGIRQ GTDTGHLNG TFLDPALIKR VEIVRGPSAL LYGSGALGGV ISYDTVDAKD LLQEGQSSGF RVFGTGGTGD HSLGLGASAF G RTENLDGI VAWSSRDRGD LRQSNGETAP NDESINNMLA KGTWQIDSAQ SLSGLVRYYN NDAREPKNPQ TVEASESSNP MV DRSTIQR DAQLSYKLAP QGNDWLNADA KIYWSEVRIN AQNTGSSGEY REQITKGARL ENRSTLFADS FASHLLTYGG EYY RQEQHP GGATTGFPQA KIDFSSGWLQ DEITLRDLPI TLLGGTRYDS YRGSSDGYKD VDADKWSSRA GMTINPTNWL MLFG SYAQA FRAPTMGEMY NDSKHFSIGR FYTNYWVPNP NLRPETNETQ EYGFGLRFDD LMLSNDALEF KASYFDTKAK DYIST TVDF AAATTMSYNV PNAKIWGWDV MTKYTTDLFS LDVAYNRTRG KDTDTGEYIS SINPDTVTST LNIPIAHSGF SVGWVG TFA DRSTHISSSY SKQPGYGVND FYVSYQGQQA LKGMTTTLVL GNAFDKEYWS PQGIPQDGRN GKIFVSYQW

UniProtKB: Outer membrane heme/hemoglobin receptor

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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

Concentration3.5 mg/mL
BufferpH: 8
Component:
ConcentrationName
0.1 MolarTris
0.15 MolarNaCl
0.01 %Dodecyl Maltoside
GridModel: Quantifoil / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: 4D-STEM / Cs: 2.7 mm / Nominal defocus max: 1.4000000000000001 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 131924
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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