|Entry||Database: EMDB / ID: EMD-3688|
|Title||Structure of a pre-catalytic spliceosome (B7 map)|
|Sample||Pre-catalytic B complex Spliceosome|
|Biological species||Saccharomyces cerevisiae (baker's yeast)|
|Method||single particle reconstruction / cryo EM / Resolution: 4 Å|
|Authors||Plaschka C / Lin P-C / Nagai K|
|Citation||Journal: Nature / Year: 2017|
Title: Structure of a pre-catalytic spliceosome.
Authors: Clemens Plaschka / Pei-Chun Lin / Kiyoshi Nagai /
Abstract: Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome's catalytic centre. Here we report the cryo-electron microscopy ...Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome's catalytic centre. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 small nuclear ribonucleoprotein particle (snRNP) associates with U4/U6.U5 tri-snRNP through the U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2. The 3' region of the U2 snRNP is flexibly attached to the SF3b-containing domain and protrudes over the concave surface of tri-snRNP, where the U1 snRNP may reside before its release from the pre-mRNA 5' splice site. The U6 ACAGAGA sequence forms a hairpin that weakly tethers the 5' splice site. The B complex proteins Prp38, Snu23 and Spp381 bind the Prp8 N-terminal domain and stabilize U6 ACAGAGA stem-pre-mRNA and Brr2-U4 small nuclear RNA interactions. These results provide important insights into the events leading to active site formation.
|Validation Report||Summary, Full report, XML, About validation report|
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_3688.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.43 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Additional map: unsharpened B7 map
-Entire Pre-catalytic B complex Spliceosome
|Entire||Name: Pre-catalytic B complex Spliceosome / Number of components: 1|
-Component #1: protein, Pre-catalytic B complex Spliceosome
|Protein||Name: Pre-catalytic B complex Spliceosome / Recombinant expression: No|
|Mass||Experimental: 2.5 MDa|
|Source||Species: Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 1.5 mg/mL / Buffer solution: Buffer pH: HEPES, 7.9; EDTA, 8.0 / pH: 7.9|
|Vitrification||Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 100 %|
Details: Grids were glow-discharged for 15 s before deposition of 3 microliter sample (~1.5 mg mL-1), and subsequently incubated for 2-3.5 s before blotting and vitrification by plunging into liquid ...Details: Grids were glow-discharged for 15 s before deposition of 3 microliter sample (~1.5 mg mL-1), and subsequently incubated for 2-3.5 s before blotting and vitrification by plunging into liquid ethane with a Vitrobot Mark III (FEI) operated at 4 degrees Celsius and 100% humidity..
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 56 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 81000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF Quantum|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Image acquisition||Number of digital images: 5115 / Sampling size: 5 µm|
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 62406 |
Details: Movies were binned once and aligned using MOTIONCORR.
|3D reconstruction||Software: RELION / Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF|
Details: A temperature factor of -80 A2 was applied (sharpened B7 map).
-Atomic model buiding
|Modeling #1||Refinement space: REAL / Overall bvalue: 80|
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