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Yorodumi- EMDB-4527: Human post-catalytic spliceosome (P complex) stalled with DHX8 K5... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-4527 | |||||||||
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Title | Human post-catalytic spliceosome (P complex) stalled with DHX8 K594A mutant, focused refinement on core | |||||||||
Map data | Human post-catalytic spliceosome (P complex) stalled with DHX8 K594A mutant, focused refinement on core | |||||||||
Sample |
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Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.0 Å | |||||||||
Authors | Fica SM / Oubridge C / Wilkinson ME / Newman AJ / Nagai K | |||||||||
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. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_4527.map.gz | 243.7 MB | EMDB map data format | |
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Header (meta data) | emd-4527-v30.xml emd-4527.xml | 16.2 KB 16.2 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_4527_fsc.xml | 14.5 KB | Display | FSC data file |
Images | emd_4527.png | 111.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-4527 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-4527 | HTTPS FTP |
-Validation report
Summary document | emd_4527_validation.pdf.gz | 287.3 KB | Display | EMDB validaton report |
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Full document | emd_4527_full_validation.pdf.gz | 286.4 KB | Display | |
Data in XML | emd_4527_validation.xml.gz | 13.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4527 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4527 | HTTPS FTP |
-Related structure data
Related structure data | 4525C 4526C 4528C 4529C 4530C 4532C 4533C 4534C 4535C 6qdvC C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_4527.map.gz / Format: CCP4 / Size: 262.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Human post-catalytic spliceosome (P complex) stalled with DHX8 K594A mutant, focused refinement on core | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.2 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Human post-catalytic P complex spliceosome
Entire | Name: Human post-catalytic P complex spliceosome |
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Components |
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-Supramolecule #1: Human post-catalytic P complex spliceosome
Supramolecule | Name: Human post-catalytic P complex spliceosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#44 |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 3.0 MDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL | ||||||||||||
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Buffer | pH: 7.9 Component:
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Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III 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
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Number real images: 6200 / Average exposure time: 10.0 sec. / Average electron dose: 53.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 135000 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |