+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3541 | ||||||||||||
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Title | Structure of a spliceosome remodeled for exon ligation | ||||||||||||
Map data | Map of the spliceosomal C-star complex resulting from focussed classification on Prp22 | ||||||||||||
Sample |
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Function / homology | Function and homology information U2-type post-spliceosomal complex / U2-type post-mRNA release spliceosomal complex / mRNA branch site recognition / spliceosomal complex disassembly / cellular bud site selection / pre-mRNA 3'-splice site binding / post-mRNA release spliceosomal complex / nuclear mRNA surveillance / generation of catalytic spliceosome for first transesterification step / cis assembly of pre-catalytic spliceosome ...U2-type post-spliceosomal complex / U2-type post-mRNA release spliceosomal complex / mRNA branch site recognition / spliceosomal complex disassembly / cellular bud site selection / pre-mRNA 3'-splice site binding / post-mRNA release spliceosomal complex / nuclear mRNA surveillance / generation of catalytic spliceosome for first transesterification step / cis assembly of pre-catalytic spliceosome / splicing factor binding / U4/U6 snRNP / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / 7-methylguanosine cap hypermethylation / Prp19 complex / snRNP binding / pICln-Sm protein complex / small nuclear ribonucleoprotein complex / pre-mRNA binding / U2-type catalytic step 1 spliceosome / SMN-Sm protein complex / spliceosomal tri-snRNP complex / mRNA cis splicing, via spliceosome / U2-type spliceosomal complex / poly(U) RNA binding / commitment complex / U2-type prespliceosome assembly / U2-type catalytic step 2 spliceosome / U4 snRNP / U2 snRNP / U1 snRNP / U2-type prespliceosome / precatalytic spliceosome / Formation of TC-NER Pre-Incision Complex / generation of catalytic spliceosome for second transesterification step / spliceosomal complex assembly / Gap-filling DNA repair synthesis and ligation in TC-NER / DNA replication origin binding / mRNA 5'-splice site recognition / protein K63-linked ubiquitination / mRNA 3'-splice site recognition / Dual incision in TC-NER / DNA replication initiation / spliceosomal tri-snRNP complex assembly / U5 snRNA binding / U5 snRNP / U2 snRNA binding / U6 snRNA binding / spliceosomal snRNP assembly / positive regulation of cell cycle / pre-mRNA intronic binding / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / nuclear periphery / positive regulation of RNA splicing / spliceosomal complex / RING-type E3 ubiquitin transferase / mRNA splicing, via spliceosome / ubiquitin-protein transferase activity / metallopeptidase activity / ubiquitin protein ligase activity / RNA helicase activity / RNA helicase / DNA repair / chromatin binding / GTPase activity / mRNA binding / chromatin / GTP binding / ATP hydrolysis activity / mitochondrion / DNA binding / RNA binding / zinc ion binding / ATP binding / identical protein binding / nucleus / metal ion binding / cytoplasm / cytosol Similarity search - Function | ||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) / Baker's yeast (brewer's yeast) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.17 Å | ||||||||||||
Authors | Fica SM / Oubridge C / Galej WP / Wilkinson ME / Newman AJ / Bai X-C / Nagai K | ||||||||||||
Funding support | United Kingdom, 3 items
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Citation | Journal: Nature / Year: 2016 Title: Cryo-EM structure of the spliceosome immediately after branching. Authors: Wojciech P Galej / Max E Wilkinson / Sebastian M Fica / Chris Oubridge / Andrew J Newman / Kiyoshi Nagai / Abstract: Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the ...Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5'-splice site is cleaved but remains close to the catalytic Mg site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5'-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2'OH. The 5'-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson-Crick interactions between the branch helix and 5'-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation. | ||||||||||||
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_3541.map.gz | 248.2 MB | EMDB map data format | |
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Header (meta data) | emd-3541-v30.xml emd-3541.xml | 64.9 KB 64.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_3541_fsc_1.xml emd_3541_fsc_2.xml | 14.5 KB 14.5 KB | Display Display | FSC data file |
Images | emd_3541_1.png emd_3541_2.png | 63.7 KB 62.6 KB | ||
Others | emd_3541_additional.map.gz | 243.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3541 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3541 | HTTPS FTP |
-Validation report
Summary document | emd_3541_validation.pdf.gz | 290.8 KB | Display | EMDB validaton report |
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Full document | emd_3541_full_validation.pdf.gz | 289.9 KB | Display | |
Data in XML | emd_3541_validation.xml.gz | 6.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3541 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3541 | HTTPS FTP |
-Related structure data
Related structure data | 5mq0MC 3539C 3542C 5mpsC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10687 (Title: Yeast C, Ci, C*, and P complex spliceosomes / Data size: 8.9 TB Data #1: Unaligned movies of C-complex spliceosome with 3' splice site AG to AC mutation (Dataset 1) [micrographs - multiframe] Data #2: Unaligned movies of C and C*-complex spliceosomes with 3' splice site AG to AdG mutation (Dataset 2) [micrographs - multiframe] Data #3: Unaligned movies of C and C*-complex spliceosomes with 3' splice site AG to AdG mutation (Dataset 3) [micrographs - multiframe] Data #4: Aligned movies of C-complex spliceosomes with cold-sensitive prp16-302 mutation, purified with Cwc25 (Dataset 4) [micrographs - multiframe] Data #5: Unaligned movies of C-complex spliceosomes with cold-sensitive prp16-302 mutation, purified with Cwc25 and incubated with ATP and Mg (Dataset 5) [micrographs - multiframe] Data #6: Unaligned movies of C, C*, and P-complex spliceosomes with dominant-negative Prp22 mutation K512A, purified with Slu7 (Dataset 6) [micrographs - multiframe] Data #7: Unaligned movies of P-complex spliceosomes with dominant-negative Prp22 mutation K512A, treated with anti-3'exon RNaseH oligo, purified in presence of Mg (Dataset 9) [micrographs - single frame] Data #8: Selected C-complex particles after polishing [picked particles - single frame - processed] Data #9: Selected P-complex particles after polishing [picked particles - single frame - processed] Data #10: Various signal subtractions for C- and P-complex spliceosomes [picked particles - single frame - processed]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_3541.map.gz / Format: CCP4 / Size: 266.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Map of the spliceosomal C-star complex resulting from focussed classification on Prp22 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.43 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: Map of the spliceosomal C-star complex resulting from...
File | emd_3541_additional.map | ||||||||||||
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Annotation | Map of the spliceosomal C-star complex resulting from focussed classification on peripheral regions of U2 snRNP | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Saccharomyces cerevisiae spliceosome. Complex C just after Prp16-...
+Supramolecule #1: Saccharomyces cerevisiae spliceosome. Complex C just after Prp16-...
+Macromolecule #1: Yeast UBC4 gene for ubiquitin-conjugating enzyme
+Macromolecule #2: 5'-EXON OF UBC4 PRE-MRNA
+Macromolecule #3: S.cerevisiae chromosome II reading frame ORF YBR230c
+Macromolecule #4: Saccharomyces cerevisiae strain T.52_2H chromosome XII sequence
+Macromolecule #5: Saccharomyces cerevisiae strain WI_C_MBSP_4 chromosome VII sequence
+Macromolecule #34: 3'-EXON OF UBC4 PRE-MRNA, BOUND BY PRP22 HELICASE
+Macromolecule #6: Pre-mRNA-splicing factor 8
+Macromolecule #7: Pre-mRNA-splicing factor SNU114
+Macromolecule #8: Pre-mRNA-splicing factor CWC22
+Macromolecule #9: Pre-mRNA-splicing factor PRP46
+Macromolecule #10: Pre-mRNA-processing protein 45
+Macromolecule #11: Pre-mRNA-splicing factor BUD31
+Macromolecule #12: Pre-mRNA-splicing factor CWC2
+Macromolecule #13: Pre-mRNA-splicing factor SLT11
+Macromolecule #14: Pre-mRNA-splicing factor CEF1
+Macromolecule #15: Pre-mRNA-splicing factor CWC15
+Macromolecule #16: Pre-mRNA-splicing factor CWC21
+Macromolecule #17: Pre-mRNA-splicing factor CLF1
+Macromolecule #18: Pre-mRNA-splicing factor SYF1,PRE-MRNA-SPLICING FACTOR SYF1
+Macromolecule #19: Pre-mRNA-splicing factor 18
+Macromolecule #20: Pre-mRNA-splicing factor SLU7
+Macromolecule #21: Pre-mRNA-processing factor 17
+Macromolecule #22: UNKNOWN PROTEIN
+Macromolecule #23: Pre-mRNA-splicing factor SYF2
+Macromolecule #24: Small nuclear ribonucleoprotein-associated protein B
+Macromolecule #25: Small nuclear ribonucleoprotein Sm D3
+Macromolecule #26: Small nuclear ribonucleoprotein E
+Macromolecule #27: Small nuclear ribonucleoprotein F
+Macromolecule #28: Small nuclear ribonucleoprotein G
+Macromolecule #29: Small nuclear ribonucleoprotein Sm D1
+Macromolecule #30: Small nuclear ribonucleoprotein Sm D2
+Macromolecule #31: Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP22
+Macromolecule #32: U2 small nuclear ribonucleoprotein A'
+Macromolecule #33: U2 small nuclear ribonucleoprotein B''
+Macromolecule #35: Pre-mRNA-splicing factor SNT309
+Macromolecule #36: Pre-mRNA-processing factor 19
+Macromolecule #37: MAGNESIUM ION
+Macromolecule #38: POTASSIUM ION
+Macromolecule #39: INOSITOL HEXAKISPHOSPHATE
+Macromolecule #40: GUANOSINE-5'-TRIPHOSPHATE
+Macromolecule #41: ZINC ION
-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:
Details: NP-40 is also called IGEPAL CA-630 | ||||||||||||||||||
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 6.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 20.0 kPa | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III Details: 3.5 microlitres sample were applied to the grid, left for 25 seconds and then blotted for 3.0-3.5 seconds before plunging.. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Quantum |
Details | GIF Quantum energy filter, 20 eV slit width |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 3596 / Average exposure time: 0.8 sec. / Average electron dose: 2.0 e/Å2 Details: Total dose: 40 electrons/Angstrom^2 over 16 seconds. 20 movie frames collected at 1.25 frames per second. |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Details | Used secondary structure restraints generated in ProSMART and LibG. |
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Refinement | Space: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 330 / Target criteria: Fourier Shell Correlation |
Output model | PDB-5mq0: |