Journal: Biomolecules / Year: 2022 Title: Structural Consequences of Deproteinating the 50S Ribosome. Authors: Daniel S D Larsson / Sandesh Kanchugal P / Maria Selmer / Abstract: Ribosomes are complex ribonucleoprotein particles. Purified 50S ribosomes subjected to high-salt wash, removing a subset of ribosomal proteins (r-proteins), were shown as competent for in vitro ...Ribosomes are complex ribonucleoprotein particles. Purified 50S ribosomes subjected to high-salt wash, removing a subset of ribosomal proteins (r-proteins), were shown as competent for in vitro assembly into functional 50S subunits. Here, we used cryo-EM to determine the structures of such LiCl core particles derived from 50S subunits. A wide range of complexes with large variations in the extent of the ordered 23S rRNA and the occupancy of r-proteins were resolved to between 2.8 Å and 9 Å resolution. Many of these particles showed high similarity to in vivo and in vitro assembly intermediates, supporting the inherent stability or metastability of these states. Similar to states in early ribosome assembly, the main class showed an ordered density for the particle base around the exit tunnel, with domain V and the 3'-half of domain IV disordered. In addition, smaller core particles were discovered, where either domain II or IV was unfolded. Our data support a multi-pathway in vitro disassembly process, similar but reverse to assembly. Dependencies between complex tertiary RNA structures and RNA-protein interactions were observed, where protein extensions dissociated before the globular domains. We observed the formation of a non-native RNA structure upon protein dissociation, demonstrating that r-proteins stabilize native RNA structures and prevent non-native interactions also after folding.
Model: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: HOLEY
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV Details: continuous carbon, 3 microL of sample, incubate for 30 seconds, blot for 3.5 seconds.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
The sample stage was tilted at 0, 15 or 30 degrees
Image recording
Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 11368 / Average exposure time: 0.77 sec. / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Startup model
Type of model: OTHER / Details: Ab-initio model in RELION
Initial angle assignment
Type: OTHER
Final angle assignment
Type: OTHER / Software - Name: RELION (ver. 3.1)
Final reconstruction
Resolution.type: BY AUTHOR / Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 21489
FSC plot (resolution estimation)
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