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- EMDB-22807: The Cryo-EM Structure of Alcohol Dehyrogenase from Yeast in compl... -

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Entry
Database: EMDB / ID: EMD-22807
TitleThe Cryo-EM Structure of Alcohol Dehyrogenase from Yeast in complex with NAD+ and Trifluoro Ethanol (TFE)
Map dataCryo-EM Structure of Alcohol Dehyrogenase from Yeast in complex with NAD+ and Pyrazole
Sample
  • Complex: Alcohol Dehydrogenase NAD+ Pyrazole complex
    • Protein or peptide: ADH1 isoform 1
  • Ligand: ZINC ION
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
  • Ligand: TRIFLUOROETHANOL
KeywordsAlcohol dehydrogenase / holo-enzyme complex / OXIDOREDUCTASE
Function / homology:
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.77 Å
AuthorsSubramanian R / Chang L / Guntupalli SR
CitationJournal: Biochemistry / Year: 2014
Title: Yeast alcohol dehydrogenase structure and catalysis.
Authors: Savarimuthu Baskar Raj / S Ramaswamy / Bryce V Plapp /
Abstract: Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (ADH1) is the constitutive enzyme that reduces acetaldehyde to ethanol during the fermentation of glucose. ADH1 is a homotetramer of subunits ...Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (ADH1) is the constitutive enzyme that reduces acetaldehyde to ethanol during the fermentation of glucose. ADH1 is a homotetramer of subunits with 347 amino acid residues. A structure for ADH1 was determined by X-ray crystallography at 2.4 Å resolution. The asymmetric unit contains four different subunits, arranged as similar dimers named AB and CD. The unit cell contains two different tetramers made up of "back-to-back" dimers, AB:AB and CD:CD. The A and C subunits in each dimer are structurally similar, with a closed conformation, bound coenzyme, and the oxygen of 2,2,2-trifluoroethanol ligated to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. In contrast, the B and D subunits have an open conformation with no bound coenzyme, and the catalytic zinc has an alternative, inverted coordination with Cys-43, Cys-153, His-66, and the carboxylate of Glu-67. The asymmetry in the dimeric subunits of the tetramer provides two structures that appear to be relevant for the catalytic mechanism. The alternative coordination of the zinc may represent an intermediate in the mechanism of displacement of the zinc-bound water with alcohol or aldehyde substrates. Substitution of Glu-67 with Gln-67 decreases the catalytic efficiency by 100-fold. Previous studies of structural modeling, evolutionary relationships, substrate specificity, chemical modification, and site-directed mutagenesis are interpreted more fully with the three-dimensional structure.
History
DepositionOct 5, 2020-
Header (metadata) releaseMar 31, 2021-
Map releaseMar 31, 2021-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kcb
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22807.png

Downloads & links

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Map

FileDownload / File: emd_22807.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM Structure of Alcohol Dehyrogenase from Yeast in complex with NAD+ and Pyrazole
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 180 pix.
= 189. Å		1.05 Å/pix.
x 180 pix.
= 189. Å		1.05 Å/pix.
x 180 pix.
= 189. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-6.826809 - 9.113795
Average (Standard dev.)0.0041356995 (±0.37798807)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 188.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z189.000189.000189.000
α/β/γ90.00090.00090.000
start NX/NY/NZ15150
NX/NY/NZ9999114
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-6.8279.1140.004

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Supplemental data

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Sample components

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Entire : Alcohol Dehydrogenase NAD+ Pyrazole complex

EntireName: Alcohol Dehydrogenase NAD+ Pyrazole complex
Components
  • Complex: Alcohol Dehydrogenase NAD+ Pyrazole complex
    • Protein or peptide: ADH1 isoform 1
  • Ligand: ZINC ION
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
  • Ligand: TRIFLUOROETHANOL

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Supramolecule #1: Alcohol Dehydrogenase NAD+ Pyrazole complex

SupramoleculeName: Alcohol Dehydrogenase NAD+ Pyrazole complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 370 KDa

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Macromolecule #1: ADH1 isoform 1

MacromoleculeName: ADH1 isoform 1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 36.759906 KDa
SequenceString: SIPETQKGVI FYESHGKLEY KDIPVPKPKA NELLINVKYS GVCHTDLHAW HGDWPLPVKL PLVGGHEGAG VVVGMGENVK GWKIGDYAG IKWLNGSCMA CEYCELGNES NCPHADLSGY THDGSFQQYA TADAVQAAHI PQGTDLAQVA PILCAGITVY K ALKSANLM ...String:
SIPETQKGVI FYESHGKLEY KDIPVPKPKA NELLINVKYS GVCHTDLHAW HGDWPLPVKL PLVGGHEGAG VVVGMGENVK GWKIGDYAG IKWLNGSCMA CEYCELGNES NCPHADLSGY THDGSFQQYA TADAVQAAHI PQGTDLAQVA PILCAGITVY K ALKSANLM AGHWVAISGA AGGLGSLAVQ YAKAMGYRVL GIDGGEGKEE LFRSIGGEVF IDFTKEKDIV GAVLKATDGG AH GVINVSV SEAAIEASTR YVRANGTTVL VGMPAGAKCC SDVFNQVVKS ISIVGSYVGN RADTREALDF FARGLVKSPI KVV GLSTLP EIYEKMEKGQ IVGRYVVDTS K

UniProtKB: UNIPROTKB: A0A6A5Q6H9

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 8 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 3 / Number of copies: 4 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM

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Macromolecule #4: TRIFLUOROETHANOL

MacromoleculeName: TRIFLUOROETHANOL / type: ligand / ID: 4 / Number of copies: 4 / Formula: ETF
Molecular weightTheoretical: 100.04 Da
Chemical component information

ChemComp-ETF:
TRIFLUOROETHANOL

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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

Concentration5 mg/mL
BufferpH: 8.2 / Component - Concentration: 50.0 mM / Component - Formula: C4H11NO3 / Component - Name: Tris
Details: Tris HCl buffer 5mM with 200mM KCl adjusted to pH 8.2.
GridModel: Quantifoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK II
DetailsPurified by Size Exclusion chromatography

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.77 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 1284904
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

5env


Chain - Chain ID: A / Chain - Residue range: 1-347 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 39.3 / Target criteria: Correlation Coefficient
Output model

PDB-7kcb:
Symmetry in Yeast Alcohol Dehydrogenase 1 -Closed Form with NAD+ and Trifluoroethanol

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