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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 dataSingle-particle cryo-EM reconstruction of horse liver alcohol dehydrogenase (sharpened)
Sample
  • Complex: Alcohol dehydrogenase from equine liver
    • Protein or peptide: Alcohol dehydrogenase E chain
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
  • Ligand: ZINC ION
Function / homology
Function and homology information


alcohol dehydrogenase activity, zinc-dependent / ethanol oxidation / NAD-retinol dehydrogenase activity / alcohol dehydrogenase / retinoic acid metabolic process / retinol metabolic process / zinc ion binding / cytosol
Similarity search - Function
Zinc-containing alcohol dehydrogenases signature. / Alcohol dehydrogenase, zinc-type, conserved site / Zinc-binding dehydrogenase / Alcohol dehydrogenase-like, C-terminal / Alcohol dehydrogenase GroES-like domain / Alcohol dehydrogenase, N-terminal / Enoylreductase / Polyketide synthase, enoylreductase domain / GroES-like superfamily / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Alcohol dehydrogenase E chain
Similarity search - Component
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.
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)
AnnotationSingle-particle cryo-EM reconstruction of horse liver alcohol dehydrogenase (sharpened)
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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Mask #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
Components
  • Complex: Alcohol dehydrogenase from equine liver
    • Protein or peptide: Alcohol dehydrogenase E chain
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
  • Ligand: ZINC ION

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Supramolecule #1: Alcohol dehydrogenase from equine liver

SupramoleculeName: Alcohol dehydrogenase from equine liver / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Lyophilized horse liver ADH purchased from Sigma Aldrich was further purified to homogeneity.
Source (natural)Organism: Equus caballus (horse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 81 KDa

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Macromolecule #1: Alcohol dehydrogenase E chain

MacromoleculeName: Alcohol dehydrogenase E chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: alcohol dehydrogenase
Source (natural)Organism: Equus caballus (horse)
Molecular weightTheoretical: 39.853273 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: STAGKVIKCK AAVLWEEKKP FSIEEVEVAP PKAHEVRIKM VATGICRSDD HVVSGTLVTP LPVIAGHEAA GIVESIGEGV TTVRPGDKV IPLFTPQCGK CRVCKHPEGN FCLKNDLSMP RGTMQDGTSR FTCRGKPIHH FLGTSTFSQY TVVDEISVAK I DAASPLEK ...String:
STAGKVIKCK AAVLWEEKKP FSIEEVEVAP PKAHEVRIKM VATGICRSDD HVVSGTLVTP LPVIAGHEAA GIVESIGEGV TTVRPGDKV IPLFTPQCGK CRVCKHPEGN FCLKNDLSMP RGTMQDGTSR FTCRGKPIHH FLGTSTFSQY TVVDEISVAK I DAASPLEK VCLIGCGFST GYGSAVKVAK VTQGSTCAVF GLGGVGLSVI MGCKAAGAAR IIGVDINKDK FAKAKEVGAT EC VNPQDYK KPIQEVLTEM SNGGVDFSFE VIGRLDTMVT ALSCCQEAYG VSVIVGVPPD SQNLSMNPML LLSGRTWKGA IFG GFKSKD SVPKLVADFM AKKFALDPLI THVLPFEKIN EGFDLLRSGE SIRTILTF

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Macromolecule #2: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 2 / Number of copies: 2 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM / Nicotinamide adenine dinucleotide

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration2.5 mg/mL
BufferpH: 8.5
Component:
ConcentrationNameFormula
20.0 mMTris
100.0 mMSodium chlorideNaClSodium chloride
1.0 mMTCEP
0.5 mMNicotinamide adenine dinucleotideNADHNicotinamide adenine dinucleotide
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / 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 horse liver ADH (Sigma Aldrich) was solubilized and further purified to homogeneity.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
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 - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-44 / Number grids imaged: 2 / Number real images: 1151 / Average exposure time: 11.0 sec. / Average electron dose: 69.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1232543
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: OTHER
Details: An ab initio model was generated from 659,662 template-picked particles using cryoSPARC and low-pass filtered to 30 A for use as an initial model for 3D refinement.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 11672
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 67
Output model

PDB-6nbb:
Horse liver alcohol dehydrogenase determined using single-particle cryo-EM at 200 keV

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