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- PDB-9cqn: Human OxyHb (C2 symmetry) obtained using the SPT Labtech chameleo... -

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Basic information

Entry
Database: PDB / ID: 9cqn
TitleHuman OxyHb (C2 symmetry) obtained using the SPT Labtech chameleon In the presence of 25 uM sodium dithionite under Al's oil
Components
  • Hemoglobin subunit alpha
  • Hemoglobin subunit beta
KeywordsOXYGEN BINDING / Hemoglobin / anaerobic / oxygen / heme
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
PROTOPORPHYRIN IX CONTAINING FE / OXYGEN MOLECULE / Hemoglobin subunit beta / Hemoglobin subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.37 Å
AuthorsCook, B.D. / Narehood, S.M. / McGuire, K.L. / Li, Y. / Tezcan, F.A. / Herzik, M.A.
Funding support United States, 3items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 30, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Hemoglobin subunit alpha
B: Hemoglobin subunit beta
C: Hemoglobin subunit alpha
D: Hemoglobin subunit beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)63,96412
Polymers61,3704
Non-polymers2,5948
Water5,224290
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Hemoglobin subunit alpha / Alpha-globin / Hemoglobin alpha chain


Mass: 14894.027 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P69905
#2: Protein Hemoglobin subunit beta / Beta-globin / Hemoglobin beta chain


Mass: 15791.067 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P68871
#3: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-OXY / OXYGEN MOLECULE


Mass: 31.999 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: O2 / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 290 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human oxyHb (C2 symmetry) determined using the SPT Labtech chameleon in the presence of 25 uM sodium dithionite under Al's oil
Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Molecular weightValue: 0.064 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
11.5441177 mMPotassium Phosphate monobasicKH2PO41
2155.17241 mMSodium ChlorideNaCl1
32.7089553 mMSodium Phosphate dibasicNa2HPO4-7H2O1
425 uMSodium DithioniteNa2S2O41
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: SPOTITON / Cryogen name: ETHANE / Humidity: 75 % / Chamber temperature: 298.15 K / Details: Samples were frozen with the SPT Labtech chameleon

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2475
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
2EPU2image acquisition
13PHENIX1.21.1_5286model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3643613
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 320799 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 85.9 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6NBC

6nbc


Accession code: 6NBC / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0034638
ELECTRON MICROSCOPYf_angle_d0.5456358
ELECTRON MICROSCOPYf_dihedral_angle_d10.466620
ELECTRON MICROSCOPYf_chiral_restr0.037688
ELECTRON MICROSCOPYf_plane_restr0.005794

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