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- EMDB-13320: Human carboxyhemoglobin bound to Staphylococcus aureus hemophore ... -

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Basic information

Entry
Database: EMDB / ID: EMD-13320
TitleHuman carboxyhemoglobin bound to Staphylococcus aureus hemophore IsdB - 1:2 complex
Map data
Sample
  • Complex: Human carboxyhemoglobin bound to Staphylococcus aureus hemophore IsdB - 1:2 complex
    • Complex: Hemoglobin
      • Protein or peptide: Hemoglobin subunit alpha
      • Protein or peptide: Hemoglobin subunit beta
    • Complex: Iron-regulated surface determinant protein B
      • Protein or peptide: Iron-regulated surface determinant protein B
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: water
KeywordsIron acquisition / Hemophore / Hemoglobin / NEAT domain / METAL TRANSPORT
Function / homology
Function and homology information


heme transmembrane transporter activity / nitric oxide transport / hemoglobin alpha binding / cellular oxidant detoxification / hemoglobin binding / haptoglobin-hemoglobin complex / renal absorption / organic acid binding / hemoglobin complex / oxygen transport ...heme transmembrane transporter activity / nitric oxide transport / hemoglobin alpha binding / cellular oxidant detoxification / hemoglobin binding / haptoglobin-hemoglobin complex / renal absorption / organic acid binding / hemoglobin complex / oxygen transport / Scavenging of heme from plasma / endocytic vesicle lumen / blood vessel diameter maintenance / hydrogen peroxide catabolic process / oxygen carrier activity / carbon dioxide transport / Late endosomal microautophagy / Heme signaling / Erythrocytes take up oxygen and release carbon dioxide / Erythrocytes take up carbon dioxide and release oxygen / response to hydrogen peroxide / Cytoprotection by HMOX1 / platelet aggregation / oxygen binding / regulation of blood pressure / Chaperone Mediated Autophagy / positive regulation of nitric oxide biosynthetic process / tertiary granule lumen / Factors involved in megakaryocyte development and platelet production / blood microparticle / ficolin-1-rich granule lumen / iron ion binding / heme binding / Neutrophil degranulation / extracellular space / extracellular exosome / extracellular region / membrane / metal ion binding / cytosol
Similarity search - Function
Iron-regulated surface determinant protein IsdB / : / Iron-regulated surface determinant protein H/B, linker domain / NEAT domain / Iron Transport-associated domain / NEAT domain profile. / NEAr Transporter domain / NEAT domain superfamily / : / YSIRK type signal peptide ...Iron-regulated surface determinant protein IsdB / : / Iron-regulated surface determinant protein H/B, linker domain / NEAT domain / Iron Transport-associated domain / NEAT domain profile. / NEAr Transporter domain / NEAT domain superfamily / : / YSIRK type signal peptide / YSIRK Gram-positive signal peptide / Hemoglobin, pi / Hemoglobin, alpha-type / Hemoglobin, beta-type / : / LPXTG cell wall anchor motif / Gram-positive cocci surface proteins LPxTG motif profile. / LPXTG cell wall anchor domain / Globin/Protoglobin / Globin domain profile. / Globin / Globin / Globin-like superfamily
Similarity search - Domain/homology
Hemoglobin subunit beta / Hemoglobin subunit alpha / Iron-regulated surface determinant protein B
Similarity search - Component
Biological speciesHomo sapiens (human) / Staphylococcus aureus subsp. aureus MW2 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsDe Bei O / Chirgadze DY / Hardwick SW / Luisi BF / Campanini B
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust200873/Z/16/Z United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Cryo-EM structures of staphylococcal IsdB bound to human hemoglobin reveal the process of heme extraction.
Authors: Omar De Bei / Marialaura Marchetti / Luca Ronda / Eleonora Gianquinto / Loretta Lazzarato / Dimitri Y Chirgadze / Steven W Hardwick / Lee R Cooper / Francesca Spyrakis / Ben F Luisi / ...Authors: Omar De Bei / Marialaura Marchetti / Luca Ronda / Eleonora Gianquinto / Loretta Lazzarato / Dimitri Y Chirgadze / Steven W Hardwick / Lee R Cooper / Francesca Spyrakis / Ben F Luisi / Barbara Campanini / Stefano Bettati /
Abstract: Iron surface determinant B (IsdB) is a hemoglobin (Hb) receptor essential for hemic iron acquisition by Staphylococcus aureus. Heme transfer to IsdB is possible from oxidized Hb (metHb), but ...Iron surface determinant B (IsdB) is a hemoglobin (Hb) receptor essential for hemic iron acquisition by Staphylococcus aureus. Heme transfer to IsdB is possible from oxidized Hb (metHb), but inefficient from Hb either bound to oxygen (oxyHb) or bound to carbon monoxide (HbCO), and encompasses a sequence of structural events that are currently poorly understood. By single-particle cryo-electron microscopy, we determined the structure of two IsdB:Hb complexes, representing key species along the heme extraction pathway. The IsdB:HbCO structure, at 2.9-Å resolution, provides a snapshot of the preextraction complex. In this early stage of IsdB:Hb interaction, the hemophore binds to the β-subunits of the Hb tetramer, exploiting a folding-upon-binding mechanism that is likely triggered by a cis/trans isomerization of Pro173. Binding of IsdB to α-subunits occurs upon dissociation of the Hb tetramer into α/β dimers. The structure of the IsdB:metHb complex reveals the final step of the extraction process, where heme transfer to IsdB is completed. The stability of the complex, both before and after heme transfer from Hb to IsdB, is influenced by isomerization of Pro173. These results greatly enhance current understanding of structural and dynamic aspects of the heme extraction mechanism by IsdB and provide insight into the interactions that stabilize the complex before the heme transfer event. This information will support future efforts to identify inhibitors of heme acquisition by S. aureus by interfering with IsdB:Hb complex formation.
History
DepositionAug 3, 2021-
Header (metadata) releaseApr 13, 2022-
Map releaseApr 13, 2022-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13320.png

Downloads & links

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Map

FileDownload / File: emd_13320.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 360 pix.
= 234.72 Å		0.65 Å/pix.
x 360 pix.
= 234.72 Å		0.65 Å/pix.
x 360 pix.
= 234.72 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.652 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.06805166 - 0.28738564
Average (Standard dev.)0.00039287598 (±0.008459965)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 234.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13320_msk_1.map
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Additional map: The map was obtained using phenix.autosharpen

Fileemd_13320_additional_1.map
AnnotationThe map was obtained using phenix.autosharpen
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Additional map: This map was obtained using the cryoSPARC tool...

Fileemd_13320_additional_2.map
AnnotationThis map was obtained using the cryoSPARC tool for the local resolution calculation
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Half map: #2

Fileemd_13320_half_map_1.map
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Half map: #1

Fileemd_13320_half_map_2.map
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Sample components

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Entire : Human carboxyhemoglobin bound to Staphylococcus aureus hemophore ...

EntireName: Human carboxyhemoglobin bound to Staphylococcus aureus hemophore IsdB - 1:2 complex
Components
  • Complex: Human carboxyhemoglobin bound to Staphylococcus aureus hemophore IsdB - 1:2 complex
    • Complex: Hemoglobin
      • Protein or peptide: Hemoglobin subunit alpha
      • Protein or peptide: Hemoglobin subunit beta
    • Complex: Iron-regulated surface determinant protein B
      • Protein or peptide: Iron-regulated surface determinant protein B
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: water

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Supramolecule #1: Human carboxyhemoglobin bound to Staphylococcus aureus hemophore ...

SupramoleculeName: Human carboxyhemoglobin bound to Staphylococcus aureus hemophore IsdB - 1:2 complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 151 KDa

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Supramolecule #2: Hemoglobin

SupramoleculeName: Hemoglobin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Iron-regulated surface determinant protein B

SupramoleculeName: Iron-regulated surface determinant protein B / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Staphylococcus aureus subsp. aureus MW2 (bacteria)

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Macromolecule #1: Hemoglobin subunit alpha

MacromoleculeName: Hemoglobin subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.150353 KDa
SequenceString:
VLSPADKTNV KAAWGKVGAH AGEYGAEALE RMFLSFPTTK TYFPHFDLSH GSAQVKGHGK KVADALTNAV AHVDDMPNAL SALSDLHAH KLRVDPVNFK LLSHCLLVTL AAHLPAEFTP AVHASLDKFL ASVSTVLTSK YR

UniProtKB: Hemoglobin subunit alpha

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Macromolecule #2: Hemoglobin subunit beta

MacromoleculeName: Hemoglobin subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.890198 KDa
SequenceString:
VHLTPEEKSA VTALWGKVNV DEVGGEALGR LLVVYPWTQR FFESFGDLST PDAVMGNPKV KAHGKKVLGA FSDGLAHLDN LKGTFATLS ELHCDKLHVD PENFRLLGNV LVCVLAHHFG KEFTPPVQAA YQKVVAGVAN ALAHKYH

UniProtKB: Hemoglobin subunit beta

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Macromolecule #3: Iron-regulated surface determinant protein B

MacromoleculeName: Iron-regulated surface determinant protein B / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus aureus subsp. aureus MW2 (bacteria)
Molecular weightTheoretical: 43.393129 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MLNQELREAI KNPAIKDKDH SAPNSRPIDF EMKKKDGTQQ FYHYASSVKP ARVIFTDSKP EIELGLQSGQ FWRKFEVYEG DKKLPIKLV SYDTVKDYAY IRFSVSNGTK AVKIVSSTHF NNKEEKYDYT LMEFAQPIYN SADKFKTEED YKAEKLLAPY K KAKTLERQ ...String:
MLNQELREAI KNPAIKDKDH SAPNSRPIDF EMKKKDGTQQ FYHYASSVKP ARVIFTDSKP EIELGLQSGQ FWRKFEVYEG DKKLPIKLV SYDTVKDYAY IRFSVSNGTK AVKIVSSTHF NNKEEKYDYT LMEFAQPIYN SADKFKTEED YKAEKLLAPY K KAKTLERQ VYELNKIQDK LPEKLKAEYK KKLEDTKKAL DEQVKSAITE FQNVQPTNEK MTDLQDTKYV VYESVENNES MM DTFVKHP IKTGMLNGKK YMVMETTNDD YWKDFMVEGQ RVRTISKDAK NNTRTIIFPY VEGKTLYDAI VKVHVKTIDY DGQ YHVRIV DKEAFTKANT DKSNKKEQQD NSAKKEATPA TPSKPTSAWS HPQFEK

UniProtKB: Iron-regulated surface determinant protein B

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Macromolecule #4: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 4 / Number of copies: 4 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 510 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration8 mg/mL
BufferpH: 7.2
Component:
ConcentrationFormulaName
10.0 mMC8H18N2O4SHEPES
8.0 mMC32H58N2O8SCHAPSO

Details: CHAPSO was added immediately before plunge freezing to overcome preferred orientation of the particles in the vitreous ice.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Pressure: 38.5035 kPa / Details: The grid was discharged on both sides.
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 2873 / Average electron dose: 39.59 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 457000
Startup modelType of model: OTHER
Details: The startup model was obtained in cryoSPARC using the Stochastic Gradient Descent (SGD) method for the ab intio reconstruction.
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 224937
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5vmm

source_name: PDB, initial_model_type: experimental model

3p5q

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7pch:
Human carboxyhemoglobin bound to Staphylococcus aureus hemophore IsdB - 1:2 complex

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