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- EMDB-2902: Cryo electron microscopy of actively translating human polysomes ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2902
TitleCryo electron microscopy of actively translating human polysomes (POST-i2 state).
Map dataPOST-i2 state of human polysomes
Sample
  • Sample: Native ribosomal complex from polysomes - POST-i2 state
  • Complex: 80S ribosomal complex
Keywordsmammalian ribosome / translation / polysome / cryo electron microscopy / elongation cycle
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.4 Å
AuthorsBehrmann E / Loerke J / Budkevich TV / Yamamoto K / Schmidt A / Penczek PA / Vos MR / Burger J / Mielke T / Scheerer P / Spahn CMT
CitationJournal: Cell / Year: 2015
Title: Structural snapshots of actively translating human ribosomes.
Authors: Elmar Behrmann / Justus Loerke / Tatyana V Budkevich / Kaori Yamamoto / Andrea Schmidt / Pawel A Penczek / Matthijn R Vos / Jörg Bürger / Thorsten Mielke / Patrick Scheerer / Christian M T Spahn /
Abstract: Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical ...Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.
History
DepositionFeb 19, 2015-
Header (metadata) releaseMar 11, 2015-
Map releaseMay 13, 2015-
UpdateMay 20, 2015-
Current statusMay 20, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

post-i2.tif

Downloads & links

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Map

FileDownload / File: emd_2902.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPOST-i2 state of human polysomes
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.89 Å/pix.
x 200 pix.
= 378. Å		1.89 Å/pix.
x 200 pix.
= 378. Å		1.89 Å/pix.
x 200 pix.
= 378. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.89 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-5.00314665 - 8.902525900000001
Average (Standard dev.)0.00313642 (±0.99049318)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 378.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.891.891.89
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z378.000378.000378.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-5.0038.9030.003

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Supplemental data

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Sample components

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Entire : Native ribosomal complex from polysomes - POST-i2 state

EntireName: Native ribosomal complex from polysomes - POST-i2 state
Components
  • Sample: Native ribosomal complex from polysomes - POST-i2 state
  • Complex: 80S ribosomal complex

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Supramolecule #1000: Native ribosomal complex from polysomes - POST-i2 state

SupramoleculeName: Native ribosomal complex from polysomes - POST-i2 state
type: sample / ID: 1000 / Details: The sample was monodisperse / Number unique components: 1
Molecular weightExperimental: 4.5 MDa / Theoretical: 4.5 MDa

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Supramolecule #1: 80S ribosomal complex

SupramoleculeName: 80S ribosomal complex / type: complex / ID: 1 / Name.synonym: 80S ribosome
Details: The sample was isolated as polysomes from the cell. Structural analysis shows that the complex contains 2 tRNAs and mRNA.
Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: HEK 293T / Location in cell: Cytoplasm
Molecular weightExperimental: 4.5 MDa / Theoretical: 4.5 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

Concentration3.5 mg/mL
BufferpH: 7.5
Details: 20 mM Hepes-KOH, pH 7.5, 100 mM KCl, 1.5 mM MgCl2, 0.5 mM spermidine, 0.04 mM spermine, 1 mM DTT
GridDetails: Quantifoil grids with additional continuous carbon support
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: FEI VITROBOT MARK II / Method: blot for 2-4 seconds before plunging

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Electron microscopy #1

Microscopy ID1
MicroscopeFEI POLARA 300
DetailsData was collected automatically with Leginon
DateAug 20, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 51282 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 205000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 115000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Electron microscopy #2

Microscopy ID2
MicroscopeFEI TITAN KRIOS
DateNov 29, 2012
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 51282 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 172000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.4 Å / Resolution method: OTHER / Software - Name: Signature, CTFFind3, Spider, SPARX
Details: Maps were calculated from 2 datasets using SSNR-weighted combination.
Number images used: 54672

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