|Entry||Database: EMDB / ID: EMD-4535|
|Title||Human post-catalytic spliceosome (P complex), focused refinement on U5 Sm|
|Sample||Human post-catalytic P complex spliceosome|
|Biological species||Homo sapiens (human)|
|Method||single particle reconstruction / cryo EM / Resolution: 3.3 Å|
|Authors||Fica SM / Oubridge C / Wilkinson ME / Newman AJ / Nagai KN|
|Citation||Journal: Science / Year: 2019|
Title: A human postcatalytic spliceosome structure reveals essential roles of metazoan factors for exon ligation.
Authors: Sebastian M Fica / Chris Oubridge / Max E Wilkinson / Andrew J Newman / Kiyoshi Nagai /
Abstract: During exon ligation, the spliceosome recognizes the 3'-splice site (3'SS) of precursor messenger RNA (pre-mRNA) through non-Watson-Crick pairing with the 5'SS and the branch adenosine, in a ...During exon ligation, the spliceosome recognizes the 3'-splice site (3'SS) of precursor messenger RNA (pre-mRNA) through non-Watson-Crick pairing with the 5'SS and the branch adenosine, in a conformation stabilized by Prp18 and Prp8. Here we present the 3.3-angstrom cryo-electron microscopy structure of a human postcatalytic spliceosome just after exon ligation. The 3'SS docks at the active site through conserved RNA interactions in the absence of Prp18. Unexpectedly, the metazoan-specific FAM32A directly bridges the 5'-exon and intron 3'SS of pre-mRNA and promotes exon ligation, as shown by functional assays. CACTIN, SDE2, and NKAP-factors implicated in alternative splicing-further stabilize the catalytic conformation of the spliceosome during exon ligation. Together these four proteins act as exon ligation factors. Our study reveals how the human spliceosome has co-opted additional proteins to modulate a conserved RNA-based mechanism for 3'SS selection and to potentially fine-tune alternative splicing at the exon ligation stage.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_4535.map.gz / Format: CCP4 / Size: 262.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.2 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Human post-catalytic P complex spliceosome
|Entire||Name: Human post-catalytic P complex spliceosome / Number of components: 1|
-Component #1: protein, Human post-catalytic P complex spliceosome
|Protein||Name: Human post-catalytic P complex spliceosome / Recombinant expression: No|
|Mass||Theoretical: 3.0 MDa|
|Source||Species: Homo sapiens (human)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 1 mg/mL / pH: 7.9|
|Vitrification||Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %|
Details: 3 uL sample was applied to the grid, left for 25s, then blotted for 2.5-3.5s and immediately plunged into liquid ethane..
-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: 60 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 130000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500.0 - 3500.0 nm|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
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