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- PDB-5gan: The overall structure of the yeast spliceosomal U4/U6.U5 tri-snRN... -
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Basic information
Entry | Database: PDB / ID: 5gan | ||||||
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Title | The overall structure of the yeast spliceosomal U4/U6.U5 tri-snRNP at 3.7 Angstrom | ||||||
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Function / homology | ![]() spliceosomal conformational changes to generate catalytic conformation / mRNA decay by 5' to 3' exoribonuclease / snoRNA splicing / maturation of 5S rRNA / snoRNA guided rRNA 2'-O-methylation / Lsm1-7-Pat1 complex / U6 snRNP / positive regulation of primary miRNA processing / positive regulation of RNA binding / box C/D sno(s)RNA 3'-end processing ...spliceosomal conformational changes to generate catalytic conformation / mRNA decay by 5' to 3' exoribonuclease / snoRNA splicing / maturation of 5S rRNA / snoRNA guided rRNA 2'-O-methylation / Lsm1-7-Pat1 complex / U6 snRNP / positive regulation of primary miRNA processing / positive regulation of RNA binding / box C/D sno(s)RNA 3'-end processing / generation of catalytic spliceosome for first transesterification step / deadenylation-dependent decapping of nuclear-transcribed mRNA / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | ||||||
Biological species | ![]() ![]() ![]() | ||||||
Method | ![]() ![]() | ||||||
![]() | Nguyen, T.H.D. / Galej, W.P. / Bai, X.C. / Oubridge, C. / Scheres, S.H.W. / Newman, A.J. / Nagai, K. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution. Authors: Thi Hoang Duong Nguyen / Wojciech P Galej / Xiao-Chen Bai / Chris Oubridge / Andrew J Newman / Sjors H W Scheres / Kiyoshi Nagai / ![]() Abstract: U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 Å resolution led ...U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 Å resolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5'-splice site during catalytic activation, forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the γ-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre. #1: ![]() Title: The architecture of the spliceosomal U4/U6.U5 tri-snRNP. Authors: Thi Hoang Duong Nguyen / Wojciech P Galej / Xiao-chen Bai / Christos G Savva / Andrew J Newman / Sjors H W Scheres / Kiyoshi Nagai / ![]() Abstract: U4/U6.U5 tri-snRNP is a 1.5-megadalton pre-assembled spliceosomal complex comprising U5 small nuclear RNA (snRNA), extensively base-paired U4/U6 snRNAs and more than 30 proteins, including the key ...U4/U6.U5 tri-snRNP is a 1.5-megadalton pre-assembled spliceosomal complex comprising U5 small nuclear RNA (snRNA), extensively base-paired U4/U6 snRNAs and more than 30 proteins, including the key components Prp8, Brr2 and Snu114. The tri-snRNP combines with a precursor messenger RNA substrate bound to U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), and transforms into a catalytically active spliceosome after extensive compositional and conformational changes triggered by unwinding of the U4 and U6 (U4/U6) snRNAs. Here we use cryo-electron microscopy single-particle reconstruction of Saccharomyces cerevisiae tri-snRNP at 5.9 Å resolution to reveal the essentially complete organization of its RNA and protein components. The single-stranded region of U4 snRNA between its 3' stem-loop and the U4/U6 snRNA stem I is loaded into the Brr2 helicase active site ready for unwinding. Snu114 and the amino-terminal domain of Prp8 position U5 snRNA to insert its loop I, which aligns the exons for splicing, into the Prp8 active site cavity. The structure provides crucial insights into the activation process and the active site of the spliceosome. | ||||||
History |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 1.8 MB | Display | ![]() |
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PDB format | ![]() | 1.4 MB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 8012MC ![]() 8006C ![]() 8007C ![]() 8008C ![]() 8009C ![]() 8010C ![]() 8011C ![]() 8013C ![]() 8014C ![]() 5gamC ![]() 5gaoC ![]() 5gapC M: map data used to model this data C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() Data size: 75.2 Data #1: Aligned particle images of the yeast U4/U6.U5 tri-snRNP [picked particles - multiframe - processed]) |
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Links
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Assembly
Deposited unit | ![]()
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Components
-RNA chain , 3 types, 3 molecules VWU
#1: RNA chain | ![]() Mass: 51186.023 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
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#2: RNA chain | ![]() Mass: 35883.176 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#19: RNA chain | ![]() Mass: 68643.344 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
-Pre-mRNA-splicing factor ... , 3 types, 3 molecules AJC
#3: Protein | Mass: 279867.469 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
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#5: Protein | Mass: 104370.133 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#28: Protein | Mass: 114132.086 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
-U4/U6 small nuclear ribonucleoprotein ... , 2 types, 2 molecules HG
#4: Protein | Mass: 52506.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
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#8: Protein | Mass: 55974.320 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
-Protein , 7 types, 8 molecules DFBxkbEK
#6: Protein | ![]() Mass: 16798.387 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() | ||||
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#7: Protein | Mass: 56382.516 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() | ||||
#9: Protein | Mass: 246470.266 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() ![]() | ||||
#10: Protein | Mass: 8528.504 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() | ||||
#11: Protein | Mass: 22426.990 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #18: Protein | | Mass: 28660.018 Da / Num. of mol.: 1 / Source method: isolated from a natural source Details: Snu66 is mostly fitted as polyAla into helices and extended polypeptides within the EM map. The authors do not believe that the sequence number is correct. Source: (natural) ![]() ![]() ![]() #20: Protein | | Mass: 13582.855 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
-Small nuclear ribonucleoprotein ... , 6 types, 12 molecules lhmjndpeqfrg
#12: Protein | Mass: 16296.798 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #13: Protein | Mass: 12876.066 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #14: Protein | Mass: 11240.139 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #15: Protein | Mass: 10385.098 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #16: Protein | Mass: 9669.945 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() #17: Protein | Mass: 8490.809 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
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-U6 snRNA-associated Sm-like protein ... , 7 types, 7 molecules 2345678
#21: Protein | Mass: 11177.888 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
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#22: Protein | Mass: 10039.262 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#23: Protein | Mass: 21298.070 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#24: Protein | Mass: 10432.954 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#25: Protein | Mass: 9406.579 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#26: Protein | Mass: 13027.045 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
#27: Protein | Mass: 12403.378 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() ![]() |
-Non-polymers , 1 types, 1 molecules ![](data/chem/img/GTP.gif)
#29: Chemical | ChemComp-GTP / ![]() |
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-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: ![]() |
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Sample preparation
Component | Name: The overall structure of the yeast spliceosomal U4/U6.U5 tri-snRNP Type: COMPLEX / Details: 30 proteins and 3 snRNAs / Entity ID: all / Source: NATURAL |
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Molecular weight | Value: 1.5 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() ![]() ![]() |
Buffer solution | pH: 7.9 |
Buffer component | Conc.: 1 mM / Name: DTT |
Specimen | Conc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied![]() ![]() |
Specimen support | Details: Grids are made of holey carbon, carbon-coated and glow discharged in N-amylamine. Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
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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![]() ![]() |
Electron lens | Mode: BRIGHT FIELD![]() ![]() |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 16 sec. / Electron dose: 38 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2477 |
EM imaging optics | Energyfilter name![]() |
Image scans | Movie frames/image: 20 / Used frames/image: 1-20 |
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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 ONLY | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 473827 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry![]() | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction![]() | Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 140155 / Num. of class averages: 1 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Space: RECIPROCAL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 3.7→3.7 Å / Cor.coef. Fo:Fc: 0.974 / SU B: 26.326 / SU ML: 0.371 / ESU R: 0.409 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: 397.886 Å2
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Refinement step | Cycle: 1 / Total: 77375 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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