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- EMDB-45822: Human OxyHb (C2 symmetry) obtained using the SPT Labtech chameleo... -

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Basic information

Entry
Database: EMDB / ID: EMD-45822
TitleHuman OxyHb (C2 symmetry) obtained using the SPT Labtech chameleon In the presence of 5 mM sodium dithionite under Al's oil
Map dataSharpened EM map of low [NaDT] OxyHb; C2 symmetry
Sample
  • Complex: Human oxyHb (C2 symmetry) determined using the SPT Labtech chameleon in the presence of 5 mM sodium dithionite under Al's oil
    • Protein or peptide: Hemoglobin subunit alpha
    • Protein or peptide: Hemoglobin subunit beta
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: OXYGEN MOLECULE
  • Ligand: water
KeywordsHemoglobin / anaerobic / oxygen / heme / OXYGEN BINDING
Function / homology
Function and homology information


nitric oxide transport / hemoglobin alpha binding / cellular oxidant detoxification / hemoglobin binding / renal absorption / haptoglobin-hemoglobin complex / organic acid binding / hemoglobin complex / oxygen transport / Scavenging of heme from plasma ...nitric oxide transport / hemoglobin alpha binding / cellular oxidant detoxification / hemoglobin binding / renal absorption / haptoglobin-hemoglobin complex / 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 / Heme signaling / Erythrocytes take up oxygen and release carbon dioxide / Erythrocytes take up carbon dioxide and release oxygen / Late endosomal microautophagy / Cytoprotection by HMOX1 / response to hydrogen peroxide / oxygen binding / regulation of blood pressure / platelet aggregation / 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
Hemoglobin, pi / Hemoglobin, alpha-type / Hemoglobin, beta-type / : / Globin/Protoglobin / Globin domain profile. / Globin / Globin / Globin-like superfamily
Similarity search - Domain/homology
Hemoglobin subunit beta / Hemoglobin subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.37 Å
AuthorsCook BD / Narehood SM / McGuire KL / Li Y / Tezcan FA / Herzik MA
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R35GM138206 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM148607-02 United States
Other privateSearle Scholars
CitationJournal: Nat Commun / Year: 2025
Title: Preparation of oxygen-sensitive proteins for high-resolution cryoEM structure determination using blot-free vitrification.
Authors: Brian D Cook / Sarah M Narehood / Kelly L McGuire / Yizhou Li / F Akif Tezcan / Mark A Herzik /
Abstract: High-quality grid preparation for single-particle cryogenic electron microscopy (cryoEM) remains a bottleneck for routinely obtaining high-resolution structures. The issues that arise from ...High-quality grid preparation for single-particle cryogenic electron microscopy (cryoEM) remains a bottleneck for routinely obtaining high-resolution structures. The issues that arise from traditional grid preparation workflows are particularly exacerbated for oxygen-sensitive proteins, including metalloproteins, whereby oxygen-induced damage and alteration of oxidation states can result in protein inactivation, denaturation, and/or aggregation. Indeed, 99% of the current structures in the EMBD were prepared aerobically and limited successes for anaerobic cryoEM grid preparation exist. Current practices for anaerobic grid preparation involve a vitrification device located in an anoxic chamber, which presents significant challenges including temperature and humidity control, optimization of freezing conditions, costs for purchase and operation, as well as accessibility. Here, we present a streamlined approach that allows for the vitrification of oxygen-sensitive proteins in reduced states using an automated blot-free grid vitrification device - the SPT Labtech chameleon. This robust workflow allows for high-resolution structure determination of dynamic, oxygen-sensitive proteins, of varying complexity and molecular weight.
History
DepositionJul 19, 2024-
Header (metadata) releaseApr 30, 2025-
Map releaseApr 30, 2025-
UpdateApr 30, 2025-
Current statusApr 30, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45822.png

Downloads & links

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Map

FileDownload / File: emd_45822.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened EM map of low [NaDT] OxyHb; C2 symmetry
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.74 Å/pix.
x 352 pix.
= 258.72 Å		0.74 Å/pix.
x 352 pix.
= 258.72 Å		0.74 Å/pix.
x 352 pix.
= 258.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.735 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.324
Minimum - Maximum-1.1207578 - 1.740738
Average (Standard dev.)0.00013769121 (±0.026681192)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 258.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45822_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened EM map of low [NaDT] OxyHb; C1 symmetry

Fileemd_45822_additional_1.map
AnnotationUnsharpened EM map of low [NaDT] OxyHb; C1 symmetry
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: phenix RESOLVE modified EM map of low [NaDT] OxyHb; C2 symmetry

Fileemd_45822_additional_2.map
Annotationphenix RESOLVE modified EM map of low [NaDT] OxyHb; C2 symmetry
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A of low [NaDT] OxyHb; C2 symmetry

Fileemd_45822_half_map_1.map
AnnotationHalf map A of low [NaDT] OxyHb; C2 symmetry
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of low [NaDT] OxyHb; C2 symmetry

Fileemd_45822_half_map_2.map
AnnotationHalf map B of low [NaDT] OxyHb; C2 symmetry
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human oxyHb (C2 symmetry) determined using the SPT Labtech chamel...

EntireName: Human oxyHb (C2 symmetry) determined using the SPT Labtech chameleon in the presence of 5 mM sodium dithionite under Al's oil
Components
  • Complex: Human oxyHb (C2 symmetry) determined using the SPT Labtech chameleon in the presence of 5 mM sodium dithionite under Al's oil
    • Protein or peptide: Hemoglobin subunit alpha
    • Protein or peptide: Hemoglobin subunit beta
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: OXYGEN MOLECULE
  • Ligand: water

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Supramolecule #1: Human oxyHb (C2 symmetry) determined using the SPT Labtech chamel...

SupramoleculeName: Human oxyHb (C2 symmetry) determined using the SPT Labtech chameleon in the presence of 5 mM sodium dithionite under Al's oil
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 64 KDa

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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: 14.894027 KDa
SequenceString:
LSPADKTNVK AAWGKVGAHA GEYGAEALER MFLSFPTTKT YFPHFDLSHG SAQVKGHGKK VADALTNAVA HVDDMPNALS ALSDLHAHK LRVDPVNFKL LSHCLLVTLA AHLPAEFTPA VHASLDKFLA SVSTVLTSKY

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: 15.791067 KDa
SequenceString:
HLTPEEKSAV TALWGKVNVD EVGGEALGRL LVVYPWTQRF FESFGDLSTP DAVMGNPKVK AHGKKVLGAF SDGLAHLDNL KGTFATLSE LHCDKLHVDP ENFRLLGNVL VCVLAHHFGK EFTPPVQAAY QKVVAGVANA LAHKYH

UniProtKB: Hemoglobin subunit beta

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Macromolecule #3: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 3 / 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 #4: OXYGEN MOLECULE

MacromoleculeName: OXYGEN MOLECULE / type: ligand / ID: 4 / Number of copies: 4 / Formula: OXY
Molecular weightTheoretical: 31.999 Da
Chemical component information

ChemComp-O2:
OXYGEN MOLECULE

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 290 / 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: 7.5
Component:
ConcentrationFormulaName
1.5441177 mMKH2PO4Potassium Phosphate monobasic
155.17241 mMNaClSodium Chloride
2.7089553 mMNa2HPO4-7H2OSodium Phosphate dibasic
5.0 mMNa2S2O4Sodium Dithionite
GridModel: Quantifoil / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 298.15 K / Instrument: SPOTITON
Details: Samples were frozen with the SPT Labtech chameleon.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
SoftwareName: EPU (ver. 2)
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1538 / Average exposure time: 5.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 165000
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: 3643613
Startup modelType of model: EMDB MAP
EMDB ID:

0407

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 220989
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6nbc


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 80.5
Output model

PDB-9cqt:
Human OxyHb (C2 symmetry) obtained using the SPT Labtech chameleon In the presence of 5 mM sodium dithionite under Al's oil

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