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Yorodumi- PDB-6nbb: Horse liver alcohol dehydrogenase determined using single-particl... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6nbb | ||||||
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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 : / all-trans-retinol dehydrogenase (NAD+) activity / alcohol dehydrogenase / retinol metabolic process / retinoic acid metabolic process / zinc ion binding / cytosol Similarity search - Function | ||||||
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
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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. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6nbb.cif.gz | 1.1 MB | Display | PDBx/mmCIF format |
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PDB format | pdb6nbb.ent.gz | 1009.1 KB | Display | PDB format |
PDBx/mmJSON format | 6nbb.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6nbb_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
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Full document | 6nbb_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 6nbb_validation.xml.gz | 188.1 KB | Display | |
Data in CIF | 6nbb_validation.cif.gz | 262.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/nb/6nbb ftp://data.pdbj.org/pub/pdb/validation_reports/nb/6nbb | HTTPS FTP |
-Related structure data
Related structure data | 0406MC 0407C 0408C 0409C 6nbcC 6nbdC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10249 (Title: Horse liver alcohol dehydrogenase movies obtained using Talos Arctica operating at 200 kV equipped with a K2 Data size: 1.3 TB Data #1: Raw, unaligned movies of horse liver alcohol dehydrogenase acquired on a Talos Arctica using a K2 direct electron detector [micrographs - multiframe]) |
-Links
-Assembly
Deposited unit |
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Number of models | 10 |
-Components
#1: Protein | 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 | #3: Chemical | ChemComp-ZN / |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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 | |||||||||||||||||||||||||
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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 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Lyophilized horse liver ADH (Sigma Aldrich) was solubilized and further purified to homogeneity. | |||||||||||||||||||||||||
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
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / 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 / Specimen holder 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 |
-Processing
Software | Name: PHENIX / Version: 1.11.1_2580: / Classification: refinement | ||||||||||||||||||||||||||||||||
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EM software |
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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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