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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 data
Samplemethemoglobin from human:
(Hemoglobin subunit ...) x 2 / ligand
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
Globin / Globin/Protoglobin / Globin-like superfamily / Haemoglobin, pi / Haemoglobin, alpha-type / Haemoglobin, beta-type
Hemoglobin subunit beta / Hemoglobin subunit alpha
Biological speciesHomo sapiens (human) / Human (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsHerzik Jr MA / Wu M / Lander GC
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.
Validation ReportPDB-ID: 6nbc

SummaryFull reportAbout validation report
History
DepositionDec 6, 2018-
Header (metadata) releaseMar 13, 2019-
Map releaseMar 13, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing 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

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)
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
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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Segmentation: #1

Fileemd_0407_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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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

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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

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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

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Sample components

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Entire methemoglobin from human

EntireName: methemoglobin from human
Details: Lyophilized human methemoglobin purchased from Sigma Aldrich
Number of components: 4

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Component #1: protein, methemoglobin from human

ProteinName: methemoglobin from human
Details: Lyophilized human methemoglobin purchased from Sigma Aldrich
Recombinant expression: No
MassTheoretical: 64 kDa
SourceSpecies: Homo sapiens (human)

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Component #2: protein, Hemoglobin subunit alpha

ProteinName: Hemoglobin subunit alpha / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 14.993159 kDa
SourceSpecies: Human (human)

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Component #3: protein, Hemoglobin subunit beta

ProteinName: Hemoglobin subunit beta / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 15.459697 kDa
SourceSpecies: Human (human)

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Component #4: ligand, PROTOPORPHYRIN IX CONTAINING FE

LigandName: PROTOPORPHYRIN IX CONTAINING FE / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.616487 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 12 mg/mL / pH: 7.5
Support filmGrids were plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.).
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 90 %
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
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 69 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 73000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 5000.0 - 16000.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1673 / Sampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 24308
Details: 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).
3D reconstructionSoftware: RELION / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Input PDB model: 4N7P
Chain ID: A

Overall bvalue: 67
Output model

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