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Open data
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Basic information
Entry | Database: PDB / ID: 3jbs | ||||||
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Title | eL6 protein from yeast 60S ribosomal subunit | ||||||
![]() | eL6 | ||||||
![]() | TRANSLATION / ribosome | ||||||
Function / homology | ![]() SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / Formation of a pool of free 40S subunits / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / L13a-mediated translational silencing of Ceruloplasmin expression / cytosolic large ribosomal subunit / cytoplasmic translation / structural constituent of ribosome / RNA binding / cytosol Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | ||||||
![]() | Passos, D.O. / Lyumkis, D. | ||||||
![]() | ![]() Title: Single-particle cryoEM analysis at near-atomic resolution from several thousand asymmetric subunits. Authors: Dario Oliveira Passos / Dmitry Lyumkis / ![]() Abstract: A single-particle cryoEM reconstruction of the large ribosomal subunit from Saccharomyces cerevisiae was obtained from a dataset of ∼75,000 particles. The gold-standard and frequency-limited ...A single-particle cryoEM reconstruction of the large ribosomal subunit from Saccharomyces cerevisiae was obtained from a dataset of ∼75,000 particles. The gold-standard and frequency-limited approaches to single-particle refinement were each independently used to determine orientation parameters for the final reconstruction. Both approaches showed similar resolution curves and nominal resolution values for the 60S dataset, estimated at 2.9 Å. The amount of over-fitting present during frequency-limited refinement was quantitatively analyzed using the high-resolution phase-randomization test, and the results showed no apparent over-fitting. The number of asymmetric subunits required to reach specific resolutions was subsequently analyzed by refining subsets of the data in an ab initio manner. With our data collection and processing strategies, sub-nanometer resolution was obtained with ∼200 asymmetric subunits (or, equivalently for the ribosomal subunit, particles). Resolutions of 5.6 Å, 4.5 Å, and 3.8 Å were reached with ∼1000, ∼1600, and ∼5000 asymmetric subunits, respectively. At these resolutions, one would expect to detect alpha-helical pitch, separation of beta-strands, and separation of Cα atoms, respectively. Using this map, together with strategies for ab initio model building and model refinement, we built a region of the ribosomal protein eL6, which was missing in previous models of the yeast ribosome. The relevance for more routine high-resolution structure determination is discussed. | ||||||
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 45.1 KB | Display | ![]() |
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PDB format | ![]() | 31.8 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 6478MC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 20000.564 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: cytosolic extract / Source: (natural) ![]() ![]() |
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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 |
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Sample preparation
Component | Name: Saccharomyces cerevisiae large 60S ribosomal subunit / Type: RIBOSOME |
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Molecular weight | Value: 2.5 MDa / Experimental value: NO |
Buffer solution | Name: elution buffer (50 mM HEPES-KOH, 100 mM KOAc, 5 mM MgOAc, 1 mM EDTA, 2 mM DTT) pH: 6.8 Details: elution buffer (50 mM HEPES-KOH, 100 mM KOAc, 5 mM MgOAc, 1 mM EDTA, 2 mM DTT) |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: 400 mesh, 1.2x1.3 micron C-flat grids, plasma-treated for 5 seconds |
Vitrification | Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temp: 77 K / Humidity: 90 % Details: A 3 uL sample was applied onto a freshly plasma-treated (6 seconds, Gatan Solarus plasma cleaner) holey carbon C-flat grid (Protochips, Inc.), allowing the sample to adsorb for 30 seconds. ...Details: A 3 uL sample was applied onto a freshly plasma-treated (6 seconds, Gatan Solarus plasma cleaner) holey carbon C-flat grid (Protochips, Inc.), allowing the sample to adsorb for 30 seconds. The sample was then plunge-frozen in liquid ethane using a manual cryo-plunger in ambient atmosphere at 4 degrees C. Method: A 3 uL sample was applied onto a freshly plasma-treated (6 seconds, Gatan Solarus plasma cleaner) holey carbon C-flat grid (Protochips, Inc.), allowing the sample to adsorb for 30 seconds. ...Method: A 3 uL sample was applied onto a freshly plasma-treated (6 seconds, Gatan Solarus plasma cleaner) holey carbon C-flat grid (Protochips, Inc.), allowing the sample to adsorb for 30 seconds. The sample was then plunge-frozen in liquid ethane using a manual cryo-plunger in ambient atmosphere at 4 degrees C. |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS / Date: Sep 2, 2014 / Details: super-resolution imaging |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 22500 X / Calibrated magnification: 38167 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm Astigmatism: Objective lens astigmatism was corrected within the Leginon software. |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 25 e/Å2 / Film or detector model: GATAN K2 (4k x 4k) |
Image scans | Num. digital images: 1833 |
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Processing
EM software |
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CTF correction | Details: each particle | ||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||
3D reconstruction | Method: projection matching / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75653 / Nominal pixel size: 1.31 Å / Actual pixel size: 1.31 Å Details: B-factor of -50 applied to final map (Single particle--Applied symmetry: C1). Symmetry type: POINT | ||||||||||||
Atomic model building | B value: 50 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: FSC / Details: REFINEMENT PROTOCOL--flexible | ||||||||||||
Atomic model building | PDB-ID: 4UJQ![]() 4ujq Pdb chain-ID: H / Accession code: 4UJQ / Source name: PDB / Type: experimental model | ||||||||||||
Refinement step | Cycle: LAST
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