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- PDB-6nbc: human methemoglobin state 1 determined using single-particle cryo... -

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Basic information

Entry
Database: PDB / ID: 6nbc
Titlehuman methemoglobin state 1 determined using single-particle cryo-EM at 200 keV
Components
  • Hemoglobin subunit alpha
  • Hemoglobin subunit beta
KeywordsOXYGEN TRANSPORT / heme-binding / hetero-4-mer / globin
Function / homology
Function and homology information


nitric oxide transport / hemoglobin binding / hemoglobin alpha binding / haptoglobin-hemoglobin complex / organic acid binding / renal absorption / cellular oxidant detoxification / oxygen transport / hemoglobin complex / endocytic vesicle lumen ...nitric oxide transport / hemoglobin binding / hemoglobin alpha binding / haptoglobin-hemoglobin complex / organic acid binding / renal absorption / cellular oxidant detoxification / oxygen transport / hemoglobin complex / endocytic vesicle lumen / positive regulation of cell death / platelet aggregation / hydrogen peroxide catabolic process / regulation of blood pressure / cytosolic small ribosomal subunit / response to hydrogen peroxide / oxygen carrier activity / bicarbonate transport / oxygen binding / receptor-mediated endocytosis / positive regulation of nitric oxide biosynthetic process / tertiary granule lumen / ficolin-1-rich granule lumen / blood microparticle / blood coagulation / iron ion binding / neutrophil degranulation / heme binding / extracellular space / extracellular exosome / membrane / extracellular region / metal ion binding / cytosol
Haemoglobin, beta-type / Haemoglobin, alpha-type / Globin / Globin/Protoglobin / Globin / Globin-like superfamily / Haemoglobin, pi / Globins / Globin-like / Orthogonal Bundle / Mainly Alpha
Hemoglobin subunit beta / Hemoglobin subunit alpha
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsHerzik Jr., M.A. / Wu, M. / Lander, G.C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)DP2EB020402 United States
CitationJournal: Nat Commun / Year: 2019
Title: High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM.
Authors: Mark A Herzik / Mengyu Wu / Gabriel C Lander /
Abstract: Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While ...Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While the Volta phase plate has enabled visualization of specimens in this size range, this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.7 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity to enable investigation of drug-target interactions. Our ~2.8 Å and ~3.2 Å structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the sub-nanometer cryo-EM structure of a sub-50 kDa protein.
Validation Report
SummaryFull reportAbout validation report
History
DepositionDec 6, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2019Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.funding_organization

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Structure visualization

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  • EMDB-0407
  • Imaged by UCSF Chimera
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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)63,3728
Polymers60,9064
Non-polymers2,4664
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Number of models4

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Components

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


Mass: 14993.159 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
Plasmid details: Obtained in lyophilized form from Sigma Aldrich
References: UniProt: P69905
#2: Protein Hemoglobin subunit beta / / Beta-globin / Hemoglobin beta chain


Mass: 15459.697 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
Plasmid details: Obtained in lyophilized form from Sigma Aldrich
References: UniProt: P68871
#3: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4

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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: methemoglobin from human / Type: COMPLEX
Details: Lyophilized human methemoglobin purchased from Sigma Aldrich
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
110 mMPhosphateNa2HPO41
2137 mMSodium chlorideNaClSodium chloride1
32.7 mMPotassium ChlorideKCL1
42 mMPotassium PhosphateKH2PO41
SpecimenConc.: 12 mg/ml
Details: Lyophilized human methemoglobin (Sigma Aldrich) was solubilized.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.).
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K
Details: Sample was manually blotted for 4-5 seconds using Whatman No. 1 filter paper immediately prior to plunge-freezing.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 73000 X / Nominal defocus max: 16000 nm / Nominal defocus min: 5000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 11 sec. / Electron dose: 69 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 1673
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 44 / Used frames/image: 1-44

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2580: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1particle selection
2Leginon3.2image acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.11.2model fitting
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignment
11RELION2.1classification
12RELION2.13D reconstruction
13PHENIX1.11.1_2580model refinement
Image processingDetails: Counting mode, 250 ms frames, exposure rate of ~1.95 e- pixel-1 s-1, total exposure of ~69 e- angstrom-2 (1.57 e- angstrom-2 frame-1).
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1232543 / Details: Difference-of-Gaussian-picked.
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24308 / Symmetry type: POINT
Atomic model buildingB value: 67 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 4N7P
Pdb chain-ID: A
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0094616
ELECTRON MICROSCOPYf_angle_d1.0036334
ELECTRON MICROSCOPYf_dihedral_angle_d7.0952616
ELECTRON MICROSCOPYf_chiral_restr0.055692
ELECTRON MICROSCOPYf_plane_restr0.011792

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