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- EMDB-13319: Human methemoglobin bound to Staphylococcus aureus hemophore IsdB -

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Basic information

Entry
Database: EMDB / ID: EMD-13319
TitleHuman methemoglobin bound to Staphylococcus aureus hemophore IsdB
Map data
Sample
  • Complex: Human methemoglobin bound to Staphylococcus aureus hemophore IsdB
    • 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
Function / homology
Function and homology information


heme transmembrane transporter activity / nitric oxide transport / hemoglobin binding / hemoglobin alpha binding / haptoglobin binding / haptoglobin-hemoglobin complex / renal absorption / organic acid binding / hemoglobin complex / oxygen transport ...heme transmembrane transporter activity / nitric oxide transport / hemoglobin binding / hemoglobin alpha binding / haptoglobin 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 / Late endosomal microautophagy / Heme signaling / carbon dioxide transport / response to hydrogen peroxide / Erythrocytes take up oxygen and release carbon dioxide / Erythrocytes take up carbon dioxide and release oxygen / Cytoprotection by HMOX1 / regulation of blood pressure / oxygen binding / platelet aggregation / Chaperone Mediated Autophagy / peroxidase activity / positive regulation of nitric oxide biosynthetic process / tertiary granule lumen / Factors involved in megakaryocyte development and platelet production / ficolin-1-rich granule lumen / blood microparticle / 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 / Hemoglobin, pi ...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 / Hemoglobin, pi / YSIRK Gram-positive signal peptide / 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) / Human (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.82 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-
UpdateApr 13, 2022-
Current statusApr 13, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13319.png

Downloads & links

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Map

FileDownload / File: emd_13319.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.978 Å
Density
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.0072532366 - 0.03123662
Average (Standard dev.)0.000105588966 (±0.0014926943)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 244.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13319_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: This map was obtained using the local resolution tool in RELION-3.0.8

Fileemd_13319_additional_1.map
AnnotationThis map was obtained using the local resolution tool in RELION-3.0.8
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: This map is the result of CTF refinement,...

Fileemd_13319_additional_2.map
AnnotationThis map is the result of CTF refinement, Bayesian polishing, and post-processing in RELION-3.0.8
Projections & Slices
AxesZYX

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Histogram

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Half map: #2

Fileemd_13319_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13319_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Human methemoglobin bound to Staphylococcus aureus hemophore IsdB

EntireName: Human methemoglobin bound to Staphylococcus aureus hemophore IsdB
Components
  • Complex: Human methemoglobin bound to Staphylococcus aureus hemophore IsdB
    • 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

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Supramolecule #1: Human methemoglobin bound to Staphylococcus aureus hemophore IsdB

SupramoleculeName: Human methemoglobin bound to Staphylococcus aureus hemophore IsdB
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 119 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)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Hemoglobin subunit alpha

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

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Macromolecule #2: Hemoglobin subunit beta

MacromoleculeName: Hemoglobin subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 15.890198 KDa
SequenceString:
VHLTPEEKSA VTALWGKVNV DEVGGEALGR LLVVYPWTQR FFESFGDLST PDAVMGNPKV KAHGKKVLGA FSDGLAHLDN LKGTFATLS ELHCDKLHVD PENFRLLGNV LVCVLAHHFG KEFTPPVQAA YQKVVAGVAN ALAHKYH

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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

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Macromolecule #4: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 4 / Number of copies: 2 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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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 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 - 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: 3051 / 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: 3.1 µm / Nominal defocus min: 1.0 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: 521822
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: OTHER
Details: The startup model was obtained in RELION using the Stochastic Gradient Descent (SGD) method for the ab intio reconstruction.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.82 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 103653
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final 3D classificationSoftware - Name: RELION (ver. 3.0.8)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

5vmm

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3p5q

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RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7pcf:
Human methemoglobin bound to Staphylococcus aureus hemophore IsdB

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