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- EMDB-0406: Single-particle cryo-EM reconstruction of horse liver alcohol deh... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-0406 | |||||||||
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Title | Single-particle cryo-EM reconstruction of horse liver alcohol dehydrogenase using 200 keV | |||||||||
![]() | Single-particle cryo-EM reconstruction of horse liver alcohol dehydrogenase (sharpened) | |||||||||
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Function / homology | ![]() : / all-trans-retinol dehydrogenase (NAD+) activity / alcohol dehydrogenase / retinol metabolic process / retinoic acid metabolic process / zinc ion binding / cytosol Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.9 Å | |||||||||
![]() | Herzik Jr MA / Wu M / Lander GC | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 176.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 22.1 KB 22.1 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 21.2 KB | Display | ![]() |
Images | ![]() | 155.6 KB | ||
Masks | ![]() | 512 MB | ![]() | |
Others | ![]() ![]() ![]() | 172.4 MB 408.2 MB 408.2 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 973.8 KB | Display | ![]() |
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Full document | ![]() | 973.3 KB | Display | |
Data in XML | ![]() | 25.5 KB | Display | |
Data in CIF | ![]() | 32.7 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6nbbMC ![]() 0407C ![]() 0408C ![]() 0409C ![]() 6nbcC ![]() 6nbdC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() 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]) |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Single-particle cryo-EM reconstruction of horse liver alcohol dehydrogenase (sharpened) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.5585 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
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Projections & Slices |
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Density Histograms |
-Additional map: Single-particle cryo-EM reconstruction of horse liver alcohol dehydrogenase...
File | emd_0406_additional.map | ||||||||||||
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Annotation | Single-particle cryo-EM reconstruction of horse liver alcohol dehydrogenase (unsharpened) | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Even half map
File | emd_0406_half_map_1.map | ||||||||||||
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Annotation | Even half map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Odd half map
File | emd_0406_half_map_2.map | ||||||||||||
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Annotation | Odd half map | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Alcohol dehydrogenase from equine liver
Entire | Name: Alcohol dehydrogenase from equine liver |
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Components |
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-Supramolecule #1: Alcohol dehydrogenase from equine liver
Supramolecule | Name: Alcohol dehydrogenase from equine liver / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 Details: Lyophilized horse liver ADH purchased from Sigma Aldrich was further purified to homogeneity. |
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Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
Molecular weight | Theoretical: 81 KDa |
-Macromolecule #1: Alcohol dehydrogenase E chain
Macromolecule | Name: Alcohol dehydrogenase E chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: alcohol dehydrogenase |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 39.853273 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: STAGKVIKCK AAVLWEEKKP FSIEEVEVAP PKAHEVRIKM VATGICRSDD HVVSGTLVTP LPVIAGHEAA GIVESIGEGV TTVRPGDKV IPLFTPQCGK CRVCKHPEGN FCLKNDLSMP RGTMQDGTSR FTCRGKPIHH FLGTSTFSQY TVVDEISVAK I DAASPLEK ...String: STAGKVIKCK AAVLWEEKKP FSIEEVEVAP PKAHEVRIKM VATGICRSDD HVVSGTLVTP LPVIAGHEAA GIVESIGEGV TTVRPGDKV IPLFTPQCGK CRVCKHPEGN FCLKNDLSMP RGTMQDGTSR FTCRGKPIHH FLGTSTFSQY TVVDEISVAK I DAASPLEK VCLIGCGFST GYGSAVKVAK VTQGSTCAVF GLGGVGLSVI MGCKAAGAAR IIGVDINKDK FAKAKEVGAT EC VNPQDYK KPIQEVLTEM SNGGVDFSFE VIGRLDTMVT ALSCCQEAYG VSVIVGVPPD SQNLSMNPML LLSGRTWKGA IFG GFKSKD SVPKLVADFM AKKFALDPLI THVLPFEKIN EGFDLLRSGE SIRTILTF |
-Macromolecule #2: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
Macromolecule | Name: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 2 / Number of copies: 2 / Formula: NAD |
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Molecular weight | Theoretical: 663.425 Da |
Chemical component information | ![]() ChemComp-NAD: |
-Macromolecule #3: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 2.5 mg/mL | |||||||||||||||
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Buffer | pH: 8.5 Component:
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Grid | Model: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER Details: Grids were plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.). | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER Details: Sample was manually blotted for 4-5 seconds using Whatman No. 1 filter paper immediately prior to plunge-freezing.. | |||||||||||||||
Details | Lyophilized horse liver ADH (Sigma Aldrich) was solubilized and further purified to homogeneity. |
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Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-44 / Number grids imaged: 2 / Number real images: 1151 / Average exposure time: 11.0 sec. / Average electron dose: 69.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 16.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 73000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |