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- EMDB-2253: negative stain single-particle reconstruction of conformation VI ... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-2253 | |||||||||
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Title | negative stain single-particle reconstruction of conformation VI of the Ltn1 E3 ubiquitin Ligase | |||||||||
![]() | conformational snapshot VI of Ltn1 | |||||||||
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![]() | conformational heterogeneity / Ltn1/Listerin / RING E3 ubiquitin ligase / translational surveillance / neurodegenerative disease | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 43.5 Å | |||||||||
![]() | Lyumkis D / Doamekpor SK / Bengtson MH / Lee JW / Toro TB / Petroski MD / Lima CD / Potter CS / Carragher B / Joazeiro CAP | |||||||||
![]() | ![]() Title: Single-particle EM reveals extensive conformational variability of the Ltn1 E3 ligase. Authors: Dmitry Lyumkis / Selom K Doamekpor / Mario H Bengtson / Joong-Won Lee / Tasha B Toro / Matthew D Petroski / Christopher D Lima / Clinton S Potter / Bridget Carragher / Claudio A P Joazeiro / ![]() Abstract: Ltn1 is a 180-kDa E3 ubiquitin ligase that associates with ribosomes and marks certain aberrant, translationally arrested nascent polypeptide chains for proteasomal degradation. In addition to its ...Ltn1 is a 180-kDa E3 ubiquitin ligase that associates with ribosomes and marks certain aberrant, translationally arrested nascent polypeptide chains for proteasomal degradation. In addition to its evolutionarily conserved large size, Ltn1 is characterized by the presence of a conserved N terminus, HEAT/ARM repeats predicted to comprise the majority of the protein, and a C-terminal catalytic RING domain, although the protein's exact structure is unknown. We used numerous single-particle EM strategies to characterize Ltn1's structure based on negative stain and vitreous ice data. Two-dimensional classifications and subsequent 3D reconstructions of electron density maps show that Ltn1 has an elongated form and presents a continuum of conformational states about two flexible hinge regions, whereas its overall architecture is reminiscent of multisubunit cullin-RING ubiquitin ligase complexes. We propose a model of Ltn1 function based on its conformational variability and flexibility that describes how these features may play a role in cotranslational protein quality control. | |||||||||
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 | ![]() | 13.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 13.8 KB 13.8 KB | Display Display | ![]() |
Images | ![]() | 52.7 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 191.3 KB | Display | ![]() |
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Full document | ![]() | 190.5 KB | Display | |
Data in XML | ![]() | 5.8 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 2248C ![]() 2249C ![]() 2250C ![]() 2251C ![]() 2252C ![]() 2254C ![]() 2255C ![]() 2256C ![]() 2257C ![]() 2258C ![]() 2259C ![]() 2260C ![]() 2261C ![]() 2262C ![]() 2263C ![]() 2264C ![]() 2265C ![]() 2266C ![]() 2267C ![]() 2268C ![]() 2269C C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | conformational snapshot VI of Ltn1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.18 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : snapshot VI of Ltn1 conformer
Entire | Name: snapshot VI of Ltn1 conformer |
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Components |
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-Supramolecule #1000: snapshot VI of Ltn1 conformer
Supramolecule | Name: snapshot VI of Ltn1 conformer / type: sample / ID: 1000 / Number unique components: 1 |
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Molecular weight | Experimental: 180 MDa / Theoretical: 180 MDa / Method: SDS-PAGE |
-Macromolecule #1: Ltn1
Macromolecule | Name: Ltn1 / type: protein_or_peptide / ID: 1 / Name.synonym: Listerin / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Experimental: 180 MDa / Theoretical: 180 MDa |
Recombinant expression | Organism: ![]() ![]() |
-Experimental details
-Structure determination
Method | negative staining |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.01 mg/mL |
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Buffer | pH: 8 / Details: 190 mM NaCl, 20 mM Tris, 1mM BME |
Staining | Type: NEGATIVE Details: 3 microliters of sample at a concentration of 0.01 mg/mL was applied to a C-flat grid with 2-micron-diameter holes overlaid by thin 1.5 nm continuous carbon. The specimen was stained with 2% ...Details: 3 microliters of sample at a concentration of 0.01 mg/mL was applied to a C-flat grid with 2-micron-diameter holes overlaid by thin 1.5 nm continuous carbon. The specimen was stained with 2% uranyl formate 3 times, then let air-dry. |
Grid | Details: c-flat grids overlaid with thin (~1.5 nm) carbon |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
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Electron microscopy
Microscope | FEI TECNAI F20 |
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Temperature | Average: 298 K |
Date | Mar 23, 2011 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 489 / Average electron dose: 15 e/Å2 |
Tilt angle max | 0 |
Electron beam | Acceleration voltage: 120 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: single-tilt room temperature / Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle min: -55 |
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
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Image processing
Details | random-conical tilt reconstruction |
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CTF correction | Details: each micrograph |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 43.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider, Appion / Number images used: 679 |
Final two d classification | Number classes: 1 |