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Yorodumi- EMDB-4525: Human post-catalytic spliceosome (P complex) stalled with DHX8 K5... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-4525 | |||||||||
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Title | Human post-catalytic spliceosome (P complex) stalled with DHX8 K594A mutant, overall map | |||||||||
Map data | Human post-catalytic spliceosome (P complex) stalled with DHX8 K594A mutant, overall map | |||||||||
Sample |
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Keywords | spliceosome / RNA / complex / SPLICING | |||||||||
Function / homology | Function and homology information regulation of homologous chromosome segregation / endoplasmic reticulum membrane organization / second spliceosomal transesterification activity / exon-exon junction subcomplex mago-y14 / negative regulation of selenocysteine incorporation / regulation of nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / post-spliceosomal complex / negative regulation of nitric-oxide synthase activity / cellular response to selenite ion / spliceosomal complex disassembly ...regulation of homologous chromosome segregation / endoplasmic reticulum membrane organization / second spliceosomal transesterification activity / exon-exon junction subcomplex mago-y14 / negative regulation of selenocysteine incorporation / regulation of nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / post-spliceosomal complex / negative regulation of nitric-oxide synthase activity / cellular response to selenite ion / spliceosomal complex disassembly / exon-exon junction complex / selenocysteine insertion sequence binding / protein exit from endoplasmic reticulum / NOSIP mediated eNOS trafficking / pre-mRNA 3'-splice site binding / granulocyte differentiation / regulation of translation at postsynapse, modulating synaptic transmission / negative regulation of toll-like receptor signaling pathway / post-mRNA release spliceosomal complex / regulation of retinoic acid receptor signaling pathway / alternative mRNA splicing, via spliceosome / U2 snRNP binding / renal system process / endonucleolytic cleavage of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / negative regulation of interleukin-8 production / U7 snRNA binding / negative regulation of excitatory postsynaptic potential / histone pre-mRNA DCP binding / 3'-5' RNA helicase activity / negative regulation of catalytic activity / U7 snRNP / negative regulation of phosphorylation / generation of catalytic spliceosome for first transesterification step / Z-decay: degradation of maternal mRNAs by zygotically expressed factors / regulation of vitamin D receptor signaling pathway / histone pre-mRNA 3'end processing complex / negative regulation of lipopolysaccharide-mediated signaling pathway / cis assembly of pre-catalytic spliceosome / SLBP independent Processing of Histone Pre-mRNAs / regulation of mRNA processing / SLBP Dependent Processing of Replication-Dependent Histone Pre-mRNAs / Deadenylation of mRNA / embryonic cranial skeleton morphogenesis / molecular sequestering activity / protein methylation / negative regulation of interferon-beta production / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / U12-type spliceosomal complex / embryonic brain development / methylosome / nuclear retinoic acid receptor binding / 7-methylguanosine cap hypermethylation / positive regulation of androgen receptor activity / poly(A) binding / M-decay: degradation of maternal mRNAs by maternally stored factors / Prp19 complex / U1 snRNP binding / mRNA 3'-end processing / ATP-dependent activity, acting on RNA / pICln-Sm protein complex / snRNP binding / sno(s)RNA-containing ribonucleoprotein complex / RNA splicing, via transesterification reactions / U2-type catalytic step 1 spliceosome / small nuclear ribonucleoprotein complex / pre-mRNA binding / C2H2 zinc finger domain binding / regulation of mRNA splicing, via spliceosome / SMN-Sm protein complex / telomerase RNA binding / spliceosomal tri-snRNP complex / telomerase holoenzyme complex / P granule / regulation of nitric oxide biosynthetic process / U2-type spliceosomal complex / U2-type precatalytic spliceosome / positive regulation by host of viral transcription / mRNA cis splicing, via spliceosome / positive regulation of vitamin D receptor signaling pathway / commitment complex / U2-type prespliceosome assembly / nuclear vitamin D receptor binding / Transport of Mature mRNA derived from an Intron-Containing Transcript / U2-type catalytic step 2 spliceosome / Notch binding / positive regulation of alpha-beta T cell differentiation / Regulation of gene expression in late stage (branching morphogenesis) pancreatic bud precursor cells / U4 snRNP / positive regulation of mRNA splicing, via spliceosome / RUNX3 regulates NOTCH signaling / U2 snRNP / RNA Polymerase II Transcription Termination / NOTCH4 Intracellular Domain Regulates Transcription / exploration behavior / U1 snRNP / ubiquitin-ubiquitin ligase activity / positive regulation of neurogenesis / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / NOTCH3 Intracellular Domain Regulates Transcription / WD40-repeat domain binding Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) / Human adenovirus 2 | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||
Authors | Fica SM / Oubridge C | |||||||||
Funding support | United Kingdom, 2 items
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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_4525.map.gz | 243.5 MB | EMDB map data format | |
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Header (meta data) | emd-4525-v30.xml emd-4525.xml | 74.2 KB 74.2 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_4525_fsc.xml | 14.5 KB | Display | FSC data file |
Images | emd_4525.png | 49.1 KB | ||
Filedesc metadata | emd-4525.cif.gz | 20 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-4525 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-4525 | HTTPS FTP |
-Validation report
Summary document | emd_4525_validation.pdf.gz | 652 KB | Display | EMDB validaton report |
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Full document | emd_4525_full_validation.pdf.gz | 651.6 KB | Display | |
Data in XML | emd_4525_validation.xml.gz | 13.8 KB | Display | |
Data in CIF | emd_4525_validation.cif.gz | 18.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4525 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4525 | HTTPS FTP |
-Related structure data
Related structure data | 6qdvMC 9fmdM 4526C 4527C 4528C 4529C 4530C 4532C 4533C 4534C 4535C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_4525.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, overall map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
+Supramolecule #1: Human post-catalytic P complex spliceosome
+Supramolecule #2: Human post-catalytic P complex spliceosome
+Supramolecule #3: ATP-dependent RNA helicase DHX8
+Supramolecule #4: MINX introns and exons
+Macromolecule #1: U2 snRNA
+Macromolecule #2: U5 snRNA
+Macromolecule #3: U6 snRNA
+Macromolecule #11: Ligated exons: MINX mRNA
+Macromolecule #15: Intron lariat: MINX RNA
+Macromolecule #4: Eukaryotic initiation factor 4A-III, N-terminally processed
+Macromolecule #5: RNA-binding protein 8A
+Macromolecule #6: Protein mago nashi homolog 2
+Macromolecule #7: Pre-mRNA-processing-splicing factor 8
+Macromolecule #8: U5 small nuclear ribonucleoprotein 200 kDa helicase
+Macromolecule #9: 116 kDa U5 small nuclear ribonucleoprotein component
+Macromolecule #10: PRKR-interacting protein 1
+Macromolecule #12: Cactin
+Macromolecule #13: Protein FAM32A
+Macromolecule #14: Pre-mRNA-splicing factor CWC22 homolog
+Macromolecule #16: Pleiotropic regulator 1
+Macromolecule #17: SNW domain-containing protein 1
+Macromolecule #18: Protein BUD31 homolog
+Macromolecule #19: Pre-mRNA-splicing factor RBM22
+Macromolecule #20: U5 small nuclear ribonucleoprotein 40 kDa protein
+Macromolecule #21: Cell division cycle 5-like protein
+Macromolecule #22: Spliceosome-associated protein CWC15 homolog
+Macromolecule #23: Serine/arginine repetitive matrix protein 2
+Macromolecule #24: Crooked neck-like protein 1
+Macromolecule #25: Pre-mRNA-splicing factor SYF1
+Macromolecule #26: Intron-binding protein aquarius
+Macromolecule #27: ATP-dependent RNA helicase DHX8
+Macromolecule #28: U2 small nuclear ribonucleoprotein A'
+Macromolecule #29: U2 small nuclear ribonucleoprotein B''
+Macromolecule #30: NF-kappa-B-activating protein
+Macromolecule #31: Small nuclear ribonucleoprotein-associated proteins B and B'
+Macromolecule #32: Pre-mRNA-splicing factor SLU7
+Macromolecule #33: Small nuclear ribonucleoprotein Sm D3
+Macromolecule #34: Small nuclear ribonucleoprotein E
+Macromolecule #35: Small nuclear ribonucleoprotein F
+Macromolecule #36: Small nuclear ribonucleoprotein G
+Macromolecule #37: Small nuclear ribonucleoprotein Sm D1
+Macromolecule #38: Peptidyl-prolyl cis-trans isomerase-like 1
+Macromolecule #39: Small nuclear ribonucleoprotein Sm D2
+Macromolecule #40: Pre-mRNA-processing factor 17
+Macromolecule #41: Pre-mRNA-splicing factor SPF27
+Macromolecule #42: Pre-mRNA-processing factor 19
+Macromolecule #43: Pre-mRNA-splicing factor SYF2
+Macromolecule #44: Replication stress response regulator SDE2
+Macromolecule #45: MAGNESIUM ION
+Macromolecule #46: POTASSIUM ION
+Macromolecule #47: ADENOSINE-5'-TRIPHOSPHATE
+Macromolecule #48: GUANOSINE-5'-TRIPHOSPHATE
+Macromolecule #49: ZINC ION
+Macromolecule #50: INOSITOL HEXAKISPHOSPHATE
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.3 mg/mL | ||||||||||||
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Buffer | pH: 7.9 Component:
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Grid | Model: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 7 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR | ||||||||||||
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 |