|Entry||Database: PDB / ID: 6nbb|
|Title||Horse liver alcohol dehydrogenase determined using single-particle cryo-EM at 200 keV|
|Components||Alcohol dehydrogenase E chain|
|Keywords||OXIDOREDUCTASE / dehydrogenase / NADH-binding / homo-2-mer|
|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
Polyketide synthase, enoylreductase domain / GroES-like superfamily / Alcohol dehydrogenase GroES-like domain / Zinc-binding dehydrogenase / NAD(P)-binding domain superfamily / Alcohol dehydrogenase, N-terminal / Alcohol dehydrogenase, C-terminal / Alcohol dehydrogenase, zinc-type, conserved site / Medium-chain alcohol dehydrogenases, catalytic domain / Quinone Oxidoreductase; Chain A, domain 1 ...Polyketide synthase, enoylreductase domain / GroES-like superfamily / Alcohol dehydrogenase GroES-like domain / Zinc-binding dehydrogenase / NAD(P)-binding domain superfamily / Alcohol dehydrogenase, N-terminal / Alcohol dehydrogenase, C-terminal / Alcohol dehydrogenase, zinc-type, conserved site / Medium-chain alcohol dehydrogenases, catalytic domain / Quinone Oxidoreductase; Chain A, domain 1 / NAD(P)-binding Rossmann-like Domain / Alpha-Beta Complex / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Alcohol dehydrogenase E chain
|Biological species||Equus caballus (horse)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å|
|Authors||Herzik Jr., M.A. / Wu, M. / Lander, G.C.|
|Funding support|| United States, 1items |
|Citation||Journal: 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.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
Downloads & links
A: Alcohol dehydrogenase E chain
B: Alcohol dehydrogenase E chain
|Number of models||10|
Mass: 39853.273 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Equus caballus (horse) / Production host: Escherichia coli (E. coli) / References: UniProt: P00327, alcohol dehydrogenase
Mass: 663.425 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Comment: NAD*YM
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: Alcohol dehydrogenase from equine liver / Type: COMPLEX|
Details: Lyophilized horse liver ADH purchased from Sigma Aldrich was further purified to homogeneity.
Entity ID: 1 / Source: RECOMBINANT
|Molecular weight||Value: 0.081 MDa / Experimental value: NO|
|Source (natural)||Organism: Equus caballus (horse)|
|Source (recombinant)||Organism: Escherichia coli (E. coli)|
|Buffer solution||pH: 8.5|
|Specimen||Conc.: 2.5 mg/ml|
Details: Lyophilized horse liver ADH (Sigma Aldrich) was solubilized and further purified to homogeneity.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
|Specimen support||Details: Grids were plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.).|
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K|
Details: Sample was manually blotted for 4-5 seconds using Whatman No. 1 filter paper immediately prior to plunge-freezing.
-Electron microscopy imaging
Model: Talos Arctica / Image courtesy: FEI Company
|Microscopy||Model: FEI TALOS ARCTICA|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 73000 X / Nominal defocus max: 16000 nm / Nominal defocus min: 5000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Average exposure time: 11 sec. / Electron dose: 69 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 1151|
|Image scans||Sampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 44 / Used frames/image: 1-44|
|Software||Name: PHENIX / Version: 1.11.1_2580: / Classification: refinement|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Particle selection||Num. of particles selected: 1232543|
|Symmetry||Point symmetry: C2 (2 fold cyclic)|
|3D reconstruction||Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11672 / Algorithm: BACK PROJECTION / Symmetry type: POINT|
|Atomic model building||B value: 67 / Protocol: FLEXIBLE FIT / Space: REAL|
|Atomic model building||PDB-ID: 2JHF|
|Refine LS restraints|
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