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- EMDB-2276: Ribosome structures to near-atomic resolution from thirty thousan... -

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Basic information

Entry
Database: EMDB / ID: EMD-2276
TitleRibosome structures to near-atomic resolution from thirty thousand cryo-EM particles
Map dataReconstruction of S.cereviseae 80S ribosome (conformation 2)
Sample
  • Sample: S.cereviseae 80S ribosome
  • Complex: 80S ribosomeEukaryotic ribosome
Keywordscryo-EM / single-particle analysis / direct electron detectors / ribosome / RELION / statistical movie processing
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsBai XC / Fernandez IS / McMullan G / Scheres SHW
CitationJournal: Elife / Year: 2013
Title: Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles.
Authors: Xiao-Chen Bai / Israel S Fernandez / Greg McMullan / Sjors H W Scheres /
Abstract: Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet ...Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet reached its full potential. Besides fundamental limits imposed by radiation damage, poor detectors and beam-induced sample movement have been shown to degrade attainable resolutions. A new generation of direct electron detectors may ameliorate both effects. Apart from exhibiting improved signal-to-noise performance, these cameras are also fast enough to follow particle movements during electron irradiation. Here, we assess the potentials of this technology for cryo-EM structure determination. Using a newly developed statistical movie processing approach to compensate for beam-induced movement, we show that ribosome reconstructions with unprecedented resolutions may be calculated from almost two orders of magnitude fewer particles than used previously. Therefore, this methodology may expand the scope of high-resolution cryo-EM to a broad range of biological specimens.DOI:http://dx.doi.org/10.7554/eLife.00461.001.
History
DepositionJan 8, 2013-
Header (metadata) releaseJan 23, 2013-
Map releaseJan 23, 2013-
UpdateJul 3, 2013-
Current statusJul 3, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2276.jpg

Downloads & links

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Map

FileDownload / File: emd_2276.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of S.cereviseae 80S ribosome (conformation 2)
Voxel sizeX=Y=Z: 1.77 Å
Density
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum-0.95164841 - 1.61312687
Average (Standard dev.)0.00287714 (±0.08923139)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-120-120-120
Dimensions240240240
Spacing240240240
CellA=B=C: 424.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.771.771.77
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z424.800424.800424.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-120-120-120
NC/NR/NS240240240
D min/max/mean-0.9521.6130.003

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Supplemental data

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Sample components

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Entire : S.cereviseae 80S ribosome

EntireName: S.cereviseae 80S ribosome
Components
  • Sample: S.cereviseae 80S ribosome
  • Complex: 80S ribosomeEukaryotic ribosome

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Supramolecule #1000: S.cereviseae 80S ribosome

SupramoleculeName: S.cereviseae 80S ribosome / type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 4.2 MDa / Theoretical: 4.2 MDa

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Supramolecule #1: 80S ribosome

SupramoleculeName: 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YAS-2488 / synonym: Baker's yeast
Molecular weightExperimental: 4.2 MDa / Theoretical: 4.2 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

Concentration0.3 mg/mL
BufferpH: 7.45
Details: 3mM Hepes-KOH, 6.6 mM Tris-acetate pH 7.2, 3 mM NH4Cl, 6.6 mM NH4-acetate, 48 mM K-acetate, 4 mM Mg-acetate, 2.4 mM DTT
GridDetails: Quantifoil grids (2/2) with 3 nm thin carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK II / Method: Blot 2.5 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 79096 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.44 µm / Nominal defocus min: 1.191 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 80 K / Max: 90 K / Average: 85 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 59,000 times magnification
DateJul 15, 2012
Image recordingCategory: CCD / Film or detector model: FEI FALCON I (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 200 / Average electron dose: 16 e/Å2
Details: Every image is the average of sixteen frames recorded by the direct electron detector
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each image
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION
Details: Use a newly developed statistical movie processing approach to compensate for beam-induced movement
Number images used: 22638
DetailsUse a newly developed statistical movie processing to compensate for beam-induced movement

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Atomic model buiding 1

Initial modelPDB ID:

3u5b
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: Rigid body. The domains were separately fitted by manual docking using program Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

3u5c
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: Rigid body. The domains were separately fitted by manual docking using program Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 3

Initial modelPDB ID:

3u5d
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: Rigid body. The domains were separately fitted by manual docking using program Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 4

Initial modelPDB ID:

3u5e
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: Rigid body. The domains were separately fitted by manual docking using program Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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