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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 / 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 speciesEquus caballus (horse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsHerzik Jr., M.A. / Wu, M. / Lander, G.C.
Funding support United States, 1items
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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionDec 6, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2019Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._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
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / cell / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB / _pdbx_audit_support.funding_organization

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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
Polymers79,7072
Non-polymers1,5886
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Number of models10

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Components

#1: Protein Alcohol dehydrogenase E chain


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
#2: Chemical ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Nicotinamide adenine dinucleotide


Mass: 663.425 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Comment: NAD*YM
#3: Chemical
ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D 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 chlorideNaClSodium chloride1
31 mMTCEP1
40.5 mMNicotinamide adenine dinucleotideNADHNicotinamide adenine dinucleotide1
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 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: 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.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: 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 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 holderCryogen: NITROGEN / 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) / Num. of grids imaged: 2 / Num. of real images: 1151
Image scansSampling size: 5 µm / 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 selectionNum. of particles selected: 1232543
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11672 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 67 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 2JHF
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0095784
ELECTRON MICROSCOPYf_angle_d0.9217818
ELECTRON MICROSCOPYf_dihedral_angle_d5.5653444
ELECTRON MICROSCOPYf_chiral_restr0.06912
ELECTRON MICROSCOPYf_plane_restr0.008988

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