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- PDB-6nbb: Horse liver alcohol dehydrogenase determined using single-particl... -

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Basic information

Entry
Database: PDB / ID: 6nbb
TitleHorse liver alcohol dehydrogenase determined using single-particle cryo-EM at 200 keV
ComponentsAlcohol dehydrogenase E chain
KeywordsOXIDOREDUCTASE / dehydrogenase / NADH-binding / homo-2-mer
Function / homologyGroES-like superfamily / Alcohol dehydrogenase, C-terminal / Zinc-containing alcohol dehydrogenases signature. / Alcohol dehydrogenase GroES-like domain / Zinc-binding dehydrogenase / NAD(P)-binding domain superfamily / Polyketide synthase, enoylreductase domain / Alcohol dehydrogenase, N-terminal / Alcohol dehydrogenase, zinc-type, conserved site / alcohol dehydrogenase activity, zinc-dependent ...GroES-like superfamily / Alcohol dehydrogenase, C-terminal / Zinc-containing alcohol dehydrogenases signature. / Alcohol dehydrogenase GroES-like domain / Zinc-binding dehydrogenase / NAD(P)-binding domain superfamily / Polyketide synthase, enoylreductase domain / Alcohol dehydrogenase, N-terminal / Alcohol dehydrogenase, zinc-type, conserved site / 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 E chain
Function and homology information
Specimen sourceEquus caballus (horse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 2.9 Å resolution
AuthorsHerzik Jr., M.A. / Wu, M. / Lander, G.C.
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
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 6, 2018 / Release: Mar 13, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Mar 13, 2019Structure modelrepositoryInitial release
1.1Mar 20, 2019Structure modelData collection / Database referencescitation / citation_author_citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Alcohol dehydrogenase E chain
B: Alcohol dehydrogenase E chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)81,2958
Polyers79,7072
Non-polymers1,5886
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Number of models10

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Components

#1: Protein/peptide Alcohol dehydrogenase E chain


Mass: 39853.273 Da / Num. of mol.: 2 / Source: (gene. exp.) Equus caballus (horse) / Production host: Escherichia coli (E. coli) / References: UniProt: P00327, alcohol dehydrogenase
#2: Chemical ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 2 / Formula: C21H27N7O14P2 / Nicotinamide adenine dinucleotide / Comment: NAD *YM
#3: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Formula: Zn / Zinc

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 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 weightValue: 0.081 MDa / Experimental value: NO
Source (natural)Organism: Equus caballus (horse)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8.5
Buffer component
IDConc.NameFormulaBuffer ID
120 mMTris1
2100 mMSodium chlorideNaCl1
31 mMTCEP1
40.5 mMNicotinamide adenine dinucleotideNADH1
SpecimenConc.: 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 supportDetails: Grids were plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.).
Grid material: GOLD / Grid mesh size: 300 / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 kelvins
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
MicroscopyMicroscope model: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 73000 / Nominal defocus max: 16000 nm / Nominal defocus min: 5000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 microns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 11 sec. / Electron dose: 69 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 2 / Number of real images: 1151
Image scansSampling size: 5 microns / Width: 3710 / Height: 3838 / Movie frames/image: 44 / Used frames/image: 1-44

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2580: / Classification: refinement
EM software
IDNameVersionCategory
2Leginon3.2image acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.11.2model fitting
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignment
12RELION2.13D reconstruction
13PHENIX1.11.1_2580model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 1232543
SymmetryPoint symmetry: C2
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 11672 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingOverall b value: 67 / Ref protocol: FLEXIBLE FIT / Ref space: REAL
Atomic model buildingPDB-ID: 2JHF
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0095784
ELECTRON MICROSCOPYf_angle_d0.9217818
ELECTRON MICROSCOPYf_dihedral_angle_d5.5653444
ELECTRON MICROSCOPYf_chiral_restr0.060912
ELECTRON MICROSCOPYf_plane_restr0.008988

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