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- EMDB-5889: Cryo-electron microscopy of human 80S ribosome -

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Basic information

Entry
Database: EMDB / ID: EMD-5889
TitleCryo-electron microscopy of human 80S ribosome
Map datahuman 80S ribosomeEukaryotic ribosome
Sample
  • Sample: human 80S ribosomeEukaryotic ribosome
  • Complex: 80S ribosomeEukaryotic ribosome
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.85 Å
AuthorsPenczek PA / Fang J / Li X / Cheng Y / Loerke J / Spahn CMT
CitationJournal: Ultramicroscopy / Year: 2014
Title: CTER-rapid estimation of CTF parameters with error assessment.
Authors: Pawel A Penczek / Jia Fang / Xueming Li / Yifan Cheng / Justus Loerke / Christian M T Spahn /
Abstract: In structural electron microscopy, the accurate estimation of the Contrast Transfer Function (CTF) parameters, particularly defocus and astigmatism, is of utmost importance for both initial ...In structural electron microscopy, the accurate estimation of the Contrast Transfer Function (CTF) parameters, particularly defocus and astigmatism, is of utmost importance for both initial evaluation of micrograph quality and for subsequent structure determination. Due to increases in the rate of data collection on modern microscopes equipped with new generation cameras, it is also important that the CTF estimation can be done rapidly and with minimal user intervention. Finally, in order to minimize the necessity for manual screening of the micrographs by a user it is necessary to provide an assessment of the errors of fitted parameters values. In this work we introduce CTER, a CTF parameters estimation method distinguished by its computational efficiency. The efficiency of the method makes it suitable for high-throughput EM data collection, and enables the use of a statistical resampling technique, bootstrap, that yields standard deviations of estimated defocus and astigmatism amplitude and angle, thus facilitating the automation of the process of screening out inferior micrograph data. Furthermore, CTER also outputs the spatial frequency limit imposed by reciprocal space aliasing of the discrete form of the CTF and the finite window size. We demonstrate the efficiency and accuracy of CTER using a data set collected on a 300kV Tecnai Polara (FEI) using the K2 Summit DED camera in super-resolution counting mode. Using CTER we obtained a structure of the 80S ribosome whose large subunit had a resolution of 4.03Å without, and 3.85Å with, inclusion of astigmatism parameters.
History
DepositionJan 22, 2014-
Header (metadata) releaseMar 19, 2014-
Map releaseJan 21, 2015-
UpdateJan 21, 2015-
Current statusJan 21, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 8.75
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 8.75
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5889.gif

Downloads & links

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Map

FileDownload / File: emd_5889.map.gz / Format: CCP4 / Size: 81.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman 80S ribosome
Voxel sizeX=Y=Z: 1.264 Å
Density
Contour LevelBy AUTHOR: 8.75 / Movie #1: 8.75
Minimum - Maximum-6.2626152 - 48.298103330000004
Average (Standard dev.)0.60599691 (±2.57134986)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 353.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2641.2641.264
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z353.920353.920353.920
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-6.26348.2980.606

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Supplemental data

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Sample components

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Entire : human 80S ribosome

EntireName: human 80S ribosomeEukaryotic ribosome
Components
  • Sample: human 80S ribosomeEukaryotic ribosome
  • Complex: 80S ribosomeEukaryotic ribosome

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Supramolecule #1000: human 80S ribosome

SupramoleculeName: human 80S ribosome / type: sample / ID: 1000 / Details: sample was monodisperse / Oligomeric state: 1 / Number unique components: 1
Molecular weightTheoretical: 4.2 MDa

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Supramolecule #1: 80S ribosome

SupramoleculeName: 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Homo sapiens (human) / synonym: human / Tissue: kidney / Cell: HEK293 / Organelle: cytosol
Molecular weightTheoretical: 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

BufferpH: 7.5
Details: 20 mM HEPES, pH 7.5, 100 mM KCl, 1.5 mM MgCl2, 0.5 mM spermidine, 0.04 mM spermine, 1 mM DTT
GridDetails: Quantifoil 2/4 Cu/Re with custom continuous carbon film
VitrificationCryogen name: ETHANE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
DateFeb 12, 2013
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 893
Details: The data was collected in movie mode at 39,000x magnification. Frames 3 to 20 were decimated twice, motion corrected with UCSHImage4, and averaged.
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Per-particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.85 Å / Resolution method: OTHER / Software - Name: SPARX
Details: The resolution reported is for the large subunit (60S) only.
Number images used: 35198

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