Journal: Mol Cell / Year: 2020 Title: Structural Insights into the Roles of Metazoan-Specific Splicing Factors in the Human Step 1 Spliceosome. Authors: Karl Bertram / Leyla El Ayoubi / Olexandr Dybkov / Dmitry E Agafonov / Cindy L Will / Klaus Hartmuth / Henning Urlaub / Berthold Kastner / Holger Stark / Reinhard Lührmann / Abstract: Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 Å core ...Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 Å core resolution and 4.5-5.7 Å at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation. It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the B to C transition. Structural comparisons with human B, C, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.
History
Deposition
Aug 2, 2020
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Header (metadata) release
Oct 14, 2020
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Map release
Oct 14, 2020
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Update
Oct 14, 2020
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Current status
Oct 14, 2020
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Entire : Human C complex Spliceosome - High resolution core
Entire
Name: Human C complex Spliceosome - High resolution core
Components
Complex: Human C complex Spliceosome - High resolution core
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Supramolecule #1: Human C complex Spliceosome - High resolution core
Supramolecule
Name: Human C complex Spliceosome - High resolution core / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#26
Source (natural)
Organism: Homo sapiens (human) / Strain: HeLa S3
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
0.1 mg/mL
Buffer
pH: 7.9 Component:
Concentration
Formula
Name
20.0 mM
HEPES-KOH
HEPES-KOH, pH 7.9
1.5 mM
MgCl2
Magnesium Chloride
150.0 mM
NaCl
Sodium chloride
Grid
Model: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK II
Details
Monodisperse particle distribution
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Electron microscopy
Microscope
FEI TITAN KRIOS
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: 2 / Number real images: 28279 / Average exposure time: 1.0 sec. / Average electron dose: 120.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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