Journal: Science / Year: 2016 Title: Molecular architecture of the Saccharomyces cerevisiae activated spliceosome. Authors: Reinhard Rauhut / Patrizia Fabrizio / Olexandr Dybkov / Klaus Hartmuth / Vladimir Pena / Ashwin Chari / Vinay Kumar / Chung-Tien Lee / Henning Urlaub / Berthold Kastner / Holger Stark / Reinhard Lührmann / Abstract: The activated spliceosome (B) is in a catalytically inactive state and is remodeled into a catalytically active machine by the RNA helicase Prp2, but the mechanism is unclear. Here, we describe a 3D ...The activated spliceosome (B) is in a catalytically inactive state and is remodeled into a catalytically active machine by the RNA helicase Prp2, but the mechanism is unclear. Here, we describe a 3D electron cryomicroscopy structure of the Saccharomyces cerevisiae B complex at 5.8-angstrom resolution. Our model reveals that in B, the catalytic U2/U6 RNA-Prp8 ribonucleoprotein core is already established, and the 5' splice site (ss) is oriented for step 1 catalysis but occluded by protein. The first-step nucleophile-the branchsite adenosine-is sequestered within the Hsh155 HEAT domain and is held 50 angstroms away from the 5'ss. Our structure suggests that Prp2 adenosine triphosphatase-mediated remodeling leads to conformational changes in Hsh155's HEAT domain that liberate the first-step reactants for catalysis.
History
Deposition
Aug 17, 2016
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Header (metadata) release
Oct 12, 2016
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Map release
Oct 12, 2016
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Update
Jul 12, 2017
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Current status
Jul 12, 2017
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Model: Quantifoil R3.5/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Spherical aberration corrector: Cs corrector with two hexapoles
Image recording
Film or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 40.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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