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

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Database: PDB / ID: 7kcb
TitleSymmetry in Yeast Alcohol Dehydrogenase 1 -Closed Form with NAD+ and Trifluoroethanol
ComponentsADH1 isoform 1
KeywordsOXIDOREDUCTASE / Alcohol dehydrogenase / holo-enzyme complex
Function and homology information
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.77 Å
AuthorsSubramanian, R. / Chang, L. / Li, Z. / Plapp, B.V.
Journal: Biochemistry / Year: 2021
Title: Cryo-Electron Microscopy Structures of Yeast Alcohol Dehydrogenase.
Authors: Sai Rohit Guntupalli / Zhuang Li / Leifu Chang / Bryce V Plapp / Ramaswamy Subramanian /
Abstract: Structures of yeast alcohol dehydrogenase determined by X-ray crystallography show that the subunits have two different conformational states in each of the two dimers that form the tetramer. ...Structures of yeast alcohol dehydrogenase determined by X-ray crystallography show that the subunits have two different conformational states in each of the two dimers that form the tetramer. Apoenzyme and holoenzyme complexes relevant to the catalytic mechanism were described, but the asymmetry led to questions about the cooperativity of the subunits in catalysis. This study used cryo-electron microscopy (cryo-EM) to provide structures for the apoenzyme, two different binary complexes with NADH, and a ternary complex with NAD and 2,2,2-trifluoroethanol. All four subunits in each of these complexes are identical, as the tetramers have 2 symmetry, suggesting that there is no preexisting asymmetry and that the subunits can be independently active. The apoenzyme and one enzyme-NADH complex have "open" conformations and the inverted coordination of the catalytic zinc with Cys-43, His-66, Glu-67, and Cys-153, whereas another enzyme-NADH complex and the ternary complex have closed conformations with the classical coordination of the zinc with Cys-43, His-66, Cys-153, and a water or the oxygen of trifluoroethanol. The conformational change involves interactions of Arg-340 with the pyrophosphate group of the coenzyme and Glu-67. The cryo-EM and X-ray crystallography studies provide structures relevant for the catalytic mechanism.
#1: Journal: 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.
DepositionOct 5, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 31, 2021Provider: repository / Type: Initial release

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Deposited unit
A: ADH1 isoform 1
B: ADH1 isoform 1
C: ADH1 isoform 1
D: ADH1 isoform 1
hetero molecules

Theoretical massNumber of molelcules
Total (without water)150,61720

  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551


#1: Protein
ADH1 isoform 1

Mass: 36759.906 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: A0A6A5Q6H9
#2: Chemical
ChemComp-ZN / ZINC ION

Mass: 65.409 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Nicotinamide adenine dinucleotide

Mass: 663.425 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: NAD*YM
#4: Chemical
ChemComp-ETF / TRIFLUOROETHANOL / 2,2,2-Trifluoroethanol

Mass: 100.040 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H3F3O / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

Experimental details


EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

Sample preparation

ComponentName: Alcohol Dehydrogenase NAD+ Pyrazole complex / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.37 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionpH: 8.2
Details: Tris HCl buffer 5mM with 200mM KCl adjusted to pH 8.2.
Buffer componentConc.: 50 mM / Name: Tris / Formula: C4H11NO3
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Purified by Size Exclusion chromatography
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)


EM software
4Gctf1.06CTF correction
7PHENIX1.17model fitting
9PHENIX1.17model refinement
13cryoSPARC23D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 2.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1284904 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 39.3 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation Coefficient
Atomic model buildingPDB-ID: 5ENV
Pdb chain-ID: A / Pdb chain residue range: 1-347
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 43.6 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.010910756
ELECTRON MICROSCOPYf_angle_d0.870814612
ELECTRON MICROSCOPYf_chiral_restr0.06341632
ELECTRON MICROSCOPYf_plane_restr0.00621864
ELECTRON MICROSCOPYf_dihedral_angle_d20.71233904

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