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- EMDB-11047: In situ cryo-electron tomography reveals layered organization of ... -

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Basic information

Entry
Database: EMDB / ID: EMD-11047
TitleIn situ cryo-electron tomography reveals layered organization of pre-ribosome maturation in nucleoli. Large Subunit Pre-Ribosome, Class 2
Map dataChlamydomonas reinhardtii Large Subunit Pre-Ribosome, Class 2
Sample
  • Complex: nucleolar small subunit processome, class 1
Biological speciesChlamydomonas reinhardtii (plant)
Methodelectron tomography / cryo EM
AuthorsErdmann PS / Klumpe S / Hou Z / Beck F / Wilfling F / Plitzko JM / Baumeister W
Funding support Germany, 1 items
OrganizationGrant numberCountry
Alexander von Humboldt Foundation Germany
CitationJournal: Nat Commun / Year: 2021
Title: In situ cryo-electron tomography reveals gradient organization of ribosome biogenesis in intact nucleoli.
Authors: Philipp S Erdmann / Zhen Hou / Sven Klumpe / Sagar Khavnekar / Florian Beck / Florian Wilfling / Jürgen M Plitzko / Wolfgang Baumeister /
Abstract: Ribosomes comprise a large (LSU) and a small subunit (SSU) which are synthesized independently in the nucleolus before being exported into the cytoplasm, where they assemble into functional ribosomes. ...Ribosomes comprise a large (LSU) and a small subunit (SSU) which are synthesized independently in the nucleolus before being exported into the cytoplasm, where they assemble into functional ribosomes. Individual maturation steps have been analyzed in detail using biochemical methods, light microscopy and conventional electron microscopy (EM). In recent years, single particle analysis (SPA) has yielded molecular resolution structures of several pre-ribosomal intermediates. It falls short, however, of revealing the spatiotemporal sequence of ribosome biogenesis in the cellular context. Here, we present our study on native nucleoli in Chlamydomonas reinhardtii, in which we follow the formation of LSU and SSU precursors by in situ cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). By combining both positional and molecular data, we reveal gradients of ribosome maturation within the granular component (GC), offering a new perspective on how the liquid-liquid-phase separation of the nucleolus supports ribosome biogenesis.
History
DepositionMay 16, 2020-
Header (metadata) releaseMay 26, 2021-
Map releaseMay 26, 2021-
UpdateOct 13, 2021-
Current statusOct 13, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.26
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.26
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11047.png

Downloads & links

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Map

FileDownload / File: emd_11047.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationChlamydomonas reinhardtii Large Subunit Pre-Ribosome, Class 2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.42 Å/pix.
x 200 pix.
= 684. Å		3.42 Å/pix.
x 200 pix.
= 684. Å		3.42 Å/pix.
x 200 pix.
= 684. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.42 Å
Density

Histogram

Histogram (log scale)
Contour LevelMovie #1: 0.26
Minimum - Maximum-0.20934671 - 0.6229189
Average (Standard dev.)-0.0036888851 (±0.061309524)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 684.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.423.423.42
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z684.000684.000684.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.2090.623-0.004

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Supplemental data

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Mask #1

Fileemd_11047_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : nucleolar small subunit processome, class 1

EntireName: nucleolar small subunit processome, class 1
Components
  • Complex: nucleolar small subunit processome, class 1

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Supramolecule #1: nucleolar small subunit processome, class 1

SupramoleculeName: nucleolar small subunit processome, class 1 / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Location in cell: nuclear

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Detailsin situ focus ion beam milled cells
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.5 e/Å2
Details: mixture of dose-symmetric, and bidirectional tilting schemes
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: RELION (ver. 3.0) / Number images used: 61
CTF correctionSoftware - Name: CTFFIND (ver. 4)
FSC plot (resolution estimation)

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