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Yorodumi- PDB-5lj5: Overall structure of the yeast spliceosome immediately after bran... -
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-Basic information
Entry | Database: PDB / ID: 5lj5 | ||||||
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Title | Overall structure of the yeast spliceosome immediately after branching. | ||||||
Components |
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Keywords | SPLICING / spliceosome / snRNP / pre-mRNA splicing / trans-esterification / lariat intermediate / complex C | ||||||
Function / homology | Function and homology information RNA exon ligation / snRNA metabolic process / snRNA modification / post-spliceosomal complex / U2-type post-mRNA release spliceosomal complex / cellular bud site selection / post-mRNA release spliceosomal complex / 3'-5' RNA helicase activity / generation of catalytic spliceosome for first transesterification step / cis assembly of pre-catalytic spliceosome ...RNA exon ligation / snRNA metabolic process / snRNA modification / post-spliceosomal complex / U2-type post-mRNA release spliceosomal complex / cellular bud site selection / post-mRNA release spliceosomal complex / 3'-5' RNA helicase activity / 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 / ATP-dependent activity, acting on RNA / 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 / RNA splicing / 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 / nucleic acid binding / RNA helicase activity / RNA helicase / DNA repair / chromatin binding / GTPase activity / mRNA binding / chromatin / GTP binding / ATP hydrolysis activity / mitochondrion / DNA binding / RNA binding / ATP binding / identical protein binding / nucleus / metal ion binding / cytoplasm / cytosol Similarity search - Function | ||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 10 Å | ||||||
Authors | Galej, W.P. / Wilkinson, M.F. / Fica, S.M. / Oubridge, C. / Newman, A.J. / Nagai, K. | ||||||
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 | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5lj5.cif.gz | 2.3 MB | Display | PDBx/mmCIF format |
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PDB format | pdb5lj5.ent.gz | 1.7 MB | Display | PDB format |
PDBx/mmJSON format | 5lj5.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5lj5_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 5lj5_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 5lj5_validation.xml.gz | 250.7 KB | Display | |
Data in CIF | 5lj5_validation.cif.gz | 445.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/lj/5lj5 ftp://data.pdbj.org/pub/pdb/validation_reports/lj/5lj5 | HTTPS FTP |
-Related structure data
Related structure data | 4057MC 4055C 4056C 4058C 4059C 5lj3C M: map data used to model this data 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
-Assembly
Deposited unit |
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1 |
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-Components
-RNA chain , 5 types, 5 molecules UEIZV
#1: RNA chain | Mass: 57444.875 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 1039023403 |
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#2: RNA chain | Mass: 5170.152 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) |
#3: RNA chain | Mass: 24109.115 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 4718 |
#4: RNA chain | Mass: 376267.406 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 536627 |
#5: RNA chain | Mass: 35883.176 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 1039022925 |
-Pre-mRNA-splicing factor ... , 14 types, 14 molecules AFCGJLMNORSTQs
#6: Protein | Mass: 279867.469 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P33334 |
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#9: Protein | Mass: 20412.477 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53854 |
#10: Protein | Mass: 114174.008 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P36048 |
#11: Protein | Mass: 28073.836 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P21374 |
#13: Protein | Mass: 50771.289 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12417 |
#15: Protein | Mass: 18484.502 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P25337 |
#16: Protein | Mass: 38486.562 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12046 |
#17: Protein | Mass: 40988.590 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38241 |
#18: Protein | Mass: 67837.773 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03654 |
#20: Protein | Mass: 15793.596 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03375 |
#21: Protein | Mass: 82555.859 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12309 |
#22: Protein | Mass: 100344.016 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04048 |
#32: Protein | Mass: 121815.234 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P15938, RNA helicase |
#34: Protein | Mass: 20741.455 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q06091 |
-Protein , 6 types, 7 molecules BDHPbkx
#7: Protein | Mass: 246470.266 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32639, RNA helicase | ||
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#8: Protein | Mass: 32371.086 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P28320 | ||
#12: Protein | Mass: 69118.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A162ERE7 | ||
#19: Protein | Mass: 19970.195 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A162GIZ3 | ||
#23: Protein | Mass: 22426.990 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40018 #35: Protein | | Mass: 16017.708 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) |
-Pre-mRNA-processing ... , 2 types, 5 molecules Ktuvw
#14: Protein | Mass: 42548.727 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P28004 |
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#33: Protein | Mass: 56629.777 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) References: UniProt: P32523, Ligases; Forming carbon-nitrogen bonds; Acid-amino-acid ligases (peptide synthases) |
-Small nuclear ribonucleoprotein ... , 6 types, 12 molecules dnepfqgrhljm
#24: Protein | Mass: 11240.139 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P43321 #25: Protein | Mass: 10385.098 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12330 #26: Protein | Mass: 9669.945 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P54999 #27: Protein | Mass: 8490.809 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40204 #28: Protein | Mass: 16296.798 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q02260 #29: Protein | Mass: 12876.066 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q06217 |
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-U2 small nuclear ribonucleoprotein ... , 2 types, 2 molecules WY
#30: Protein | Mass: 27232.252 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08963 |
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#31: Protein | Mass: 12850.944 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40567 |
-Non-polymers , 3 types, 10 molecules
#36: Chemical | #37: Chemical | ChemComp-ZN / #38: Chemical | ChemComp-GTP / | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Spliceosome immediately after branching / Type: COMPLEX Details: Splicing extract was prepared from Prp18-HA or Slu7-TAPS yeast strains. An in vitro transcribed yeast UBC4 pre-mRNA substrate (with 2 x MS2 bacteriophage coat protein-binding stem loops at ...Details: Splicing extract was prepared from Prp18-HA or Slu7-TAPS yeast strains. An in vitro transcribed yeast UBC4 pre-mRNA substrate (with 2 x MS2 bacteriophage coat protein-binding stem loops at the 5' end and with the 3'-splice site sequence UAGAG mutated to UACAC) was pre-bound to an MS2-maltose binding protein fusion protein. This substrate-protein complex was added to the splicing extract. The splicing reaction proceeded through the first step but the second step was blocked by the 3' splice site mutation. Substrate-bound spliceosomes from the splicing extract were purified on amylose resin and eluted with maltose. Subsequently the spliceosomes were captured on anti-HA-agarose (for Prp18-HA-tagged) or streptactin resin (for Slu7-TAPS tagged) and eluted with HA peptide or desthiobiotin, respectively. Purified spliceosomes were then dialysed against 20 mM HEPES KOH pH 7.8, 75 mM KCl, 0.25 mM EDTA Entity ID: #1-#35 / Source: NATURAL | ||||||||||||||||||||||||||||||
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Molecular weight | Value: 2 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||||||||||||||||||||
Buffer solution | pH: 7.8 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K Details: 3 microlitres sample were applied to the grid, left for 30 seconds and then blotted for 2.5-3.0 seconds before plunging. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 35714 X / Nominal defocus max: 4000 nm / Nominal defocus min: 500 nm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 0.8 sec. / Electron dose: 2 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 2213 Details: Total dose: 40 electrons/Angstrom^2 over 16 seconds. 20 movie frames collected at 1.25 frames per second. |
EM imaging optics | Energyfilter name: GIF Quantum |
Image scans | Movie frames/image: 20 / Used frames/image: 1-20 |
-Processing
Software | Name: REFMAC / Version: 5.8.0124 / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 15872 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 10 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 15872 / Num. of class averages: 1 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: RECIPROCAL Details: Used secondary structure restraints generated in ProSMART and LibG. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 3.8→274.56 Å / Cor.coef. Fo:Fc: 0.954 / SU B: 27.594 / SU ML: 0.398 / ESU R: 0.522 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
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Solvent computation | Solvent model: PARAMETERS FOR MASK CACLULATION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 180.1 Å2
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Refinement step | Cycle: 1 / Total: 63166 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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