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- EMDB-6478: Single-particle cryo-electron microscopy reconstruction of the eu... -

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Basic information

Entry
Database: EMDB / ID: EMD-6478
TitleSingle-particle cryo-electron microscopy reconstruction of the eukaryotic large ribosomal subunit
Map dataSingle-particle cryo-EM reconstruction of the eukaryotic large 60S ribosomal subunit
Sample
  • Sample: Saccharomyces cerevisiae large 60S ribosomal subunit
  • Complex: 60S large ribosomal subunit
Keywordsribosome / translation
Function / homology
Function and homology information


cytoplasmic translation / cytosolic large ribosomal subunit / ribosomal large subunit assembly / structural constituent of ribosome / RNA binding / cytosol
Similarity search - Function
Ribosomal protein L6e signature. / Ribosomal Protein L6, KOW domain / Ribosomal protein L6e / 60S ribosomal protein L6E / Translation protein SH3-like domain superfamily / Ribosomal protein L2, domain 2
Similarity search - Domain/homology
60S ribosomal protein L6-A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsPassos DO / Lyumkis D
CitationJournal: J Struct Biol / Year: 2015
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.
History
DepositionOct 5, 2015-
Header (metadata) releaseOct 21, 2015-
Map releaseOct 21, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jbs
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jbs
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3jbs
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol

Downloads & links

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Map

FileDownload / File: emd_6478.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle-particle cryo-EM reconstruction of the eukaryotic large 60S ribosomal subunit
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 320 pix.
= 419.2 Å
1.31 Å/pix.
x 320 pix.
= 419.2 Å
1.31 Å/pix.
x 320 pix.
= 419.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.04220856 - 0.09491237
Average (Standard dev.)-0.00044745 (±0.00324659)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 419.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z419.200419.200419.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0420.095-0.000

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Supplemental data

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Sample components

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Entire : Saccharomyces cerevisiae large 60S ribosomal subunit

EntireName: Saccharomyces cerevisiae large 60S ribosomal subunit
Components
  • Sample: Saccharomyces cerevisiae large 60S ribosomal subunit
  • Complex: 60S large ribosomal subunit

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Supramolecule #1000: Saccharomyces cerevisiae large 60S ribosomal subunit

SupramoleculeName: Saccharomyces cerevisiae large 60S ribosomal subunit / type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 2.5 MDa

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Supramolecule #1: 60S large ribosomal subunit

SupramoleculeName: 60S large ribosomal subunit / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-eukaryote: LSU 60S
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast) / Strain: BY4741 / synonym: yeast / Location in cell: cytosol
Molecular weightTheoretical: 2.5 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6.8
Details: 50 mM HEPES-KOH, 100 mM KOAc, 5 mM MgOAc, 1 mM EDTA, 2 mM DTT
GridDetails: 400 mesh, 1.2x1.3 micron C-flat grids, plasma-treated for 5 seconds
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER
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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 38167 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected within the Leginon software.
Detailssuper-resolution imaging
DateSep 2, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 1833 / Average electron dose: 25 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: OTHER / Software - Name: Frealign / Details: B-factor of -50 applied to final map / Number images used: 75653

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Atomic model buiding 1

Initial modelPDB ID:

4ujq
PDB Unreleased entry


Chain - Chain ID: H
SoftwareName: Chimera, Rosetta
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: FSC
Output model

PDB-3jbs:
eL6 protein from yeast 60S ribosomal subunit

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