[English] 日本語
Yorodumi- PDB-5lj3: Structure of the core of the yeast spliceosome immediately after ... -
+Open data
-Basic information
Entry | Database: PDB / ID: 5lj3 | ||||||
---|---|---|---|---|---|---|---|
Title | Structure of the core of the yeast spliceosome immediately after branching | ||||||
Components |
| ||||||
Keywords | SPLICING / spliceosome / snRNP / pre-mRNA splicing / trans-esterification / lariat intermediate / complex C | ||||||
Function / homology | Function and homology information post-spliceosomal complex / U2-type post-mRNA release spliceosomal complex / cellular bud site selection / post-mRNA release spliceosomal complex / U4/U6 snRNP / cis assembly of pre-catalytic spliceosome / generation of catalytic spliceosome for first transesterification step / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / splicing factor binding / 7-methylguanosine cap hypermethylation ...post-spliceosomal complex / U2-type post-mRNA release spliceosomal complex / cellular bud site selection / post-mRNA release spliceosomal complex / U4/U6 snRNP / cis assembly of pre-catalytic spliceosome / generation of catalytic spliceosome for first transesterification step / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / splicing factor binding / 7-methylguanosine cap hypermethylation / pre-mRNA binding / U2-type catalytic step 1 spliceosome / pICln-Sm protein complex / Prp19 complex / spliceosomal tri-snRNP complex / U4 snRNP / small nuclear ribonucleoprotein complex / SMN-Sm protein complex / mRNA cis splicing, via spliceosome / U2-type spliceosomal complex / U2-type prespliceosome assembly / commitment complex / U2-type catalytic step 2 spliceosome / U2 snRNP / poly(U) RNA binding / U1 snRNP / U2-type prespliceosome / precatalytic spliceosome / spliceosomal complex assembly / Dual incision in TC-NER / DNA replication origin binding / generation of catalytic spliceosome for second transesterification step / Gap-filling DNA repair synthesis and ligation in TC-NER / mRNA 3'-splice site recognition / mRNA 5'-splice site recognition / DNA replication initiation / spliceosomal tri-snRNP complex assembly / U5 snRNA binding / U5 snRNP / spliceosomal snRNP assembly / U2 snRNA binding / U6 snRNA binding / pre-mRNA intronic binding / positive regulation of cell cycle / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / positive regulation of RNA splicing / RNA splicing / spliceosomal complex / mRNA splicing, via spliceosome / metallopeptidase activity / cell cycle / mRNA binding / GTPase activity / chromatin binding / chromatin / GTP binding / DNA binding / RNA binding / metal ion binding / nucleus / cytosol / cytoplasm Similarity search - Function | ||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 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 |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5lj3.cif.gz | 1.7 MB | Display | PDBx/mmCIF format |
---|---|---|---|---|
PDB format | pdb5lj3.ent.gz | 1.3 MB | Display | PDB format |
PDBx/mmJSON format | 5lj3.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/lj/5lj3 ftp://data.pdbj.org/pub/pdb/validation_reports/lj/5lj3 | HTTPS FTP |
---|
-Related structure data
Related structure data | 4055MC 4056C 4057C 4058C 4059C 5lj5C M: map data used to model this data C: citing same article (ref.) |
---|---|
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 |
|
---|---|
1 |
|
-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: 1039022920 |
---|---|
#2: RNA chain | Mass: 5170.152 Da / Num. of mol.: 1 / Source method: isolated from a natural source Details: This RNA was produced from an in vitro transcribed yeast UBC4 pre-mRNA with two stem-loops added at the 5' end for binding by the MS2 bacteriophage coat protein. This pre-mRNA is added to a ...Details: This RNA was produced from an in vitro transcribed yeast UBC4 pre-mRNA with two stem-loops added at the 5' end for binding by the MS2 bacteriophage coat protein. This pre-mRNA is added to a yeast splicing extract and the first step of splicing yields the 5' exon and the lariat intron-3' exon intermediate, which both remain associated with the spliceosome. 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 Details: This RNA was produced from an in vitro transcribed yeast UBC4 pre-mRNA with two stem-loops added at the 5' end for binding by the MS2 bacteriophage coat protein. This pre-mRNA is added to a ...Details: This RNA was produced from an in vitro transcribed yeast UBC4 pre-mRNA with two stem-loops added at the 5' end for binding by the MS2 bacteriophage coat protein. This pre-mRNA is added to a yeast splicing extract and the first step of splicing yields the 5' exon and the lariat intron-3' exon intermediate (this RNA), which both remain associated with the spliceosome. 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 ... , 6 types, 6 molecules AFCLNR
#6: Protein | Mass: 279867.469 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P33334 |
---|---|
#8: Protein | Mass: 20412.477 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P53854 |
#9: Protein | Mass: 114174.008 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P36048 |
#14: 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: 40988.590 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P38241 |
#19: Protein | Mass: 15793.596 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: Q03375 |
-Protein , 12 types, 13 molecules DGHJKMOPSTbkx
#7: Protein | Mass: 32371.086 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P28320 | ||
---|---|---|---|
#10: Protein | Mass: 28120.896 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A165UGV0, UniProt: P21374*PLUS | ||
#11: 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 | ||
#12: Protein | Mass: 50801.316 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A162HKW4, UniProt: Q12417*PLUS | ||
#13: 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 | ||
#15: Protein | Mass: 38458.508 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A162HWE4, UniProt: Q12046*PLUS | ||
#17: Protein | Mass: 67819.789 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A165TLN2, UniProt: Q03654*PLUS | ||
#18: 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, UniProt: Q03772*PLUS | ||
#20: Protein | Mass: 82532.867 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A165TU20, UniProt: Q12309*PLUS | ||
#21: Protein | Mass: 100356.141 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A165VVY6, UniProt: Q04048*PLUS | ||
#22: Protein | Mass: 22426.990 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P40018 #31: Protein | | Mass: 11251.861 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) |
-Small nuclear ribonucleoprotein ... , 6 types, 12 molecules dnepfqgrhljm
#23: Protein | Mass: 11240.139 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P43321 #24: Protein | Mass: 10385.098 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: Q12330 #25: 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 #26: Protein | Mass: 8490.809 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P40204 #27: Protein | Mass: 16296.798 Da / Num. of mol.: 2 / Source method: isolated from a natural source Details: MKLVNFLKKLRNEQVTIELKNGTTVWGTLQSVSPQMNAILTDVKLTLPQPRLNKLNSNGIAMASLYLTGGQQPTASDNIA SLQYINIRGNTIRQIILPDSLNLDSLLVDQKQLNSLRRSGQIANDPSKKRRRDFGAPANKRPRRGL Source: (natural) Saccharomyces cerevisiae / References: UniProt: Q02260 #28: Protein | Mass: 12876.066 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: Q06217 |
---|
-U2 small nuclear ribonucleoprotein ... , 2 types, 2 molecules WY
#29: 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 |
---|---|
#30: Protein | Mass: 12850.944 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P40567 |
-Non-polymers , 3 types, 10 molecules
#32: Chemical | #33: Chemical | ChemComp-ZN / #34: Chemical | ChemComp-GTP / | |
---|
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
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-#31 / Source: NATURAL | ||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Molecular weight | Value: 2 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||||||||||||||||||||
Buffer solution | pH: 7.8 | ||||||||||||||||||||||||||||||
Buffer component |
| ||||||||||||||||||||||||||||||
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 |
---|---|
Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / 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 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
EM software |
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 248000 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93106 / 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
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Solvent computation | Solvent model: PARAMETERS FOR MASK CACLULATION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 180.1 Å2
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement step | Cycle: 1 / Total: 63166 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
|