Journal: 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
Deposition
May 16, 2020
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Header (metadata) release
May 26, 2021
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Map release
May 26, 2021
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Update
Oct 13, 2021
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Current status
Oct 13, 2021
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Entire : nucleolar small subunit processome, class 1
Entire
Name: 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
Supramolecule
Name: 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
Method
cryo EM
Processing
electron tomography
Aggregation state
particle
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Sample preparation
Buffer
pH: 7.4
Vitrification
Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details
in situ focus ion beam milled cells
Sectioning
Other: NO SECTIONING
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film 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 beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Final reconstruction
Algorithm: BACK PROJECTION / Software - Name: RELION (ver. 3.0) / Number images used: 61
CTF correction
Software - Name: CTFFIND (ver. 4)
FSC plot (resolution estimation)
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