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- EMDB-0407: Single-particle cryo-EM reconstruction of human methemoglobin usi... -

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Basic information

Entry
Database: EMDB / ID: EMD-0407
TitleSingle-particle cryo-EM reconstruction of human methemoglobin using 200 keV, state 1
Map dataSingle-particle cryo-EM reconstruction of methemoglobin state 1 (sharpened)
Sample
  • Complex: methemoglobin from human
    • Protein or peptide: Hemoglobin subunit alpha
    • Protein or peptide: Hemoglobin subunit beta
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
Keywordsheme-binding / hetero-4-mer / globin / OXYGEN TRANSPORT
Function / homology
Function and homology information


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 ...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 / Cytoprotection by HMOX1 / response to hydrogen peroxide / 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
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: 2.8 Å
AuthorsHerzik Jr MA / Wu M
Funding support United States, 1 items
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.
History
DepositionDec 6, 2018-
Header (metadata) releaseMar 13, 2019-
Map releaseMar 13, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nbc
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0407.png

Downloads & links

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Map

FileDownload / File: emd_0407.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle-particle cryo-EM reconstruction of methemoglobin state 1 (sharpened)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.56 Å/pix.
x 256 pix.
= 142.848 Å		0.56 Å/pix.
x 256 pix.
= 142.848 Å		0.56 Å/pix.
x 256 pix.
= 142.848 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.558 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.08468923 - 0.15800197
Average (Standard dev.)0.00027734737 (±0.0074136793)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 142.848 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.5580.5580.558
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z142.848142.848142.848
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0850.1580.000

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Supplemental data

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Mask #1

Fileemd_0407_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Single-particle cryo-EM reconstruction of methemoglobin state 1 (unsharpened)...

Fileemd_0407_additional.map
AnnotationSingle-particle cryo-EM reconstruction of methemoglobin state 1 (unsharpened)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Odd half map

Fileemd_0407_half_map_1.map
AnnotationOdd half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Even half map

Fileemd_0407_half_map_2.map
AnnotationEven half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : methemoglobin from human

EntireName: methemoglobin from human
Components
  • Complex: methemoglobin from human
    • Protein or peptide: Hemoglobin subunit alpha
    • Protein or peptide: Hemoglobin subunit beta
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE

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Supramolecule #1: methemoglobin from human

SupramoleculeName: methemoglobin from human / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Lyophilized human methemoglobin purchased from Sigma Aldrich
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: 14.993159 KDa
SequenceString:
VLSPADKTNV KAAWGKVGAH AGEYGAEALE RMFLSFPTTK TYFPHFDLSH GSAQVKGHGK KVADALTNAV AHVDDMPNAL SALSDLHAH KLRVDPVNFK LLSHCLLVTL AAHLPAEFTP AVHASLDKFL ASVSTVLTSK Y

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: 15.459697 KDa
SequenceString:
VHLTPEEKSA VTALWGKVNV DEVGGEALGR LLVVYPWTQR FFESFGDLST PDAVMGNPKV KAHGKKVLGA FSDGLAHLDN LKGTFATLS ELHCDKLHVD PENFRLLGNV LVCVLAHHFG KEFTPPVQAA YQKVVAGVAN ALAH

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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration12 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMNa2HPO4Phosphate
137.0 mMNaClSodium chloride
2.7 mMKCLPotassium Chloride
2.0 mMKH2PO4Potassium Phosphate
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: OTHER
Details: Grids were plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.).
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
Details: Sample was manually blotted for 4-5 seconds using Whatman No. 1 filter paper immediately prior to plunge-freezing..
DetailsLyophilized human methemoglobin (Sigma Aldrich) was solubilized.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-44 / Number grids imaged: 2 / Number real images: 1673 / Average exposure time: 11.0 sec. / Average electron dose: 69.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 16.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 73000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

DetailsCounting 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).
Particle selectionNumber selected: 1232543 / Details: Difference-of-Gaussian-picked.
Startup modelType of model: OTHER
Details: An ab initio model was generated from 160,169 template-picked particles using cryoSPARC and low-pass filtered to 20 A for use as an initial model for 3D refinement.
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 24308
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4n7p


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 67
Output model

PDB-6nbc:
human methemoglobin state 1 determined using single-particle cryo-EM at 200 keV

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