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- EMDB-0406: Single-particle cryo-EM reconstruction of horse liver alcohol deh... -

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Basic information

Entry
Database: EMDB / ID: EMD-0406
TitleSingle-particle cryo-EM reconstruction of horse liver alcohol dehydrogenase using 200 keV
Map data
SampleAlcohol dehydrogenase from equine liver:
Alcohol dehydrogenase E chain / (ligand) x 2
Function / homology
Function and homology information


alcohol dehydrogenase activity, zinc-dependent / alcohol dehydrogenase / ethanol oxidation / retinol dehydrogenase activity / retinoic acid metabolic process / retinol metabolic process / zinc ion binding / cytosol
Alcohol dehydrogenase, zinc-type, conserved site / GroES-like superfamily / Alcohol dehydrogenase, C-terminal / Alcohol dehydrogenase, N-terminal / Polyketide synthase, enoylreductase domain / NAD(P)-binding domain superfamily
Alcohol dehydrogenase E chain
Biological speciesEquus caballus (horse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 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: 6nbb

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: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nbb
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0406.map.gz / Format: CCP4 / Size: 190.1 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 368 pix.
= 205.528 Å
0.56 Å/pix.
x 368 pix.
= 205.528 Å
0.56 Å/pix.
x 368 pix.
= 205.528 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.5585 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.03750041 - 0.06748688
Average (Standard dev.)-0.00003268129 (±0.0033607704)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin727272
Dimensions368368368
Spacing368368368
CellA=B=C: 205.528 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.55850.55850.5585
M x/y/z368368368
origin x/y/z0.0000.0000.000
length x/y/z205.528205.528205.528
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS727272
NC/NR/NS368368368
D min/max/mean-0.0380.067-0.000

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Supplemental data

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Segmentation: #1

Fileemd_0406_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Single-particle cryo-EM reconstruction of horse liver alcohol dehydrogenase...

Fileemd_0406_additional.map
AnnotationSingle-particle cryo-EM reconstruction of horse liver alcohol dehydrogenase (unsharpened)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Even half map

Fileemd_0406_half_map_1.map
AnnotationEven half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Odd half map

Fileemd_0406_half_map_2.map
AnnotationOdd half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Alcohol dehydrogenase from equine liver

EntireName: Alcohol dehydrogenase from equine liver
Details: Lyophilized horse liver ADH purchased from Sigma Aldrich was further purified to homogeneity.
Number of components: 4

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Component #1: protein, Alcohol dehydrogenase from equine liver

ProteinName: Alcohol dehydrogenase from equine liver
Details: Lyophilized horse liver ADH purchased from Sigma Aldrich was further purified to homogeneity.
Recombinant expression: No
MassTheoretical: 81 kDa
SourceSpecies: Equus caballus (horse)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Alcohol dehydrogenase E chain

ProteinName: Alcohol dehydrogenase E chain / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 39.853273 kDa
SourceSpecies: Equus caballus (horse)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: ligand, NICOTINAMIDE-ADENINE-DINUCLEOTIDE

LigandName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.663425 kDa

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Component #4: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2.5 mg/mL / pH: 8.5
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: 1151 / Sampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 11672
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / Resolution: 2.9 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: 2JHF
Overall bvalue: 67
Output model

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