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- EMDB-8007: The closed state of the yeast spliceosomal U4/U6.U5 tri-snRNP at ... -

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Basic information

Entry
Database: EMDB / ID: EMD-8007
TitleThe closed state of the yeast spliceosomal U4/U6.U5 tri-snRNP at 4.3 Angstrom
Map data
Sampletri-snRNP complex with 30 proteins and 3 snRNA:
(nucleic-acidNucleic acid) x 3 / Prp8 / Prp4 / Prp6 / Dib1 / Prp31 / Prp3 / Brr2 / unknown proteins / SmB ...(nucleic-acidNucleic acid) x 3 / Prp8 / Prp4 / Prp6 / Dib1 / Prp31 / Prp3 / Brr2 / unknown proteins / SmB / U4 SmD1 / SmD2 / SmD3 / SmE / SmF / SmG / Snu66 / U5 SmE / U5 SmD1 / Snu13NHP2L1 / LSm2 / LSm3 / LSm4 / LSm5 / LSm6 / LSm7 / LSm8 / Snu114
Biological speciesSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsNguyen THD / Galej WP / Bai X-C / Oubridge C / Newman AJ / Scheres SHW / Nagai K
CitationJournal: Nature / Year: 2016
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.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionDec 14, 2015-
Header (metadata) releaseJan 20, 2016-
Map releaseMar 30, 2016-
UpdateAug 30, 2017-
Current statusAug 30, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.032
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.032
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8007.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.43 Å/pix.
x 380 pix.
= 543.4 Å
1.43 Å/pix.
x 380 pix.
= 543.4 Å
1.43 Å/pix.
x 380 pix.
= 543.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.43 Å
Density
Contour LevelBy AUTHOR: 0.032 / Movie #1: 0.032
Minimum - Maximum-0.0643664 - 0.16928972
Average (Standard dev.)0.00025029454 (±0.006761632)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 543.39996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.431.431.43
M x/y/z380380380
origin x/y/z0.0000.0000.000
length x/y/z543.400543.400543.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-190-190-190
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS380380380
D min/max/mean-0.0640.1690.000

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Supplemental data

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Sample components

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Entire tri-snRNP complex with 30 proteins and 3 snRNA

EntireName: tri-snRNP complex with 30 proteins and 3 snRNA / Number of components: 31

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Component #1: protein, tri-snRNP complex with 30 proteins and 3 snRNA

ProteinName: tri-snRNP complex with 30 proteins and 3 snRNA / Recombinant expression: No
MassTheoretical: 1.5 MDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #2: nucleic-acid, U4 snRNA

nucleic acidName: U4 snRNAU4 spliceosomal RNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
AUCCUUAUGC ACGGGAAAUA CGCAUAUCAG UGAGGAUUCG UCCGAGAUUG UGUUUUUGCU GGUUGAAAUU UAAUUAUAAA CCAGACCGUC UCCUCAUGGU CAAUUCGGUG UUCGCUUUUG AAUACUUCAA GACUAUGUAG GGAAUUUUUG GAAUACCUUU
MassTheoretical: 39.813488 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #3: nucleic-acid, U6 snRNA

nucleic acidName: U6 snRNAU6 spliceosomal RNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
GUUCGCGAAG UAACCCUUCG UGGACAUUUG GUCAAUUUGA AACAAUACAG AGAUGAUCAG CAGUUCCCCU GCAUAAGGAU GAACCGUUUU ACAAAGAGAU UUAUUUCGUU UU
MassTheoretical: 25.635123 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #4: protein, Prp8

ProteinName: Prp8 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 255.921422 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #5: protein, Prp4

ProteinName: Prp4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 39.953031 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #6: protein, Prp6

ProteinName: Prp6 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 84.644094 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #7: protein, Dib1

ProteinName: Dib1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 16.480982 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #8: protein, Prp31

ProteinName: Prp31 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 47.128652 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #9: protein, Prp3

ProteinName: Prp3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 37.87025 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #10: protein, Brr2

ProteinName: Brr2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 202.6655 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #11: protein, unknown proteins

ProteinName: unknown proteins / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 8.528504 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #12: protein, SmB

ProteinName: SmB / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 9.183853 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #13: protein, U4 SmD1

ProteinName: U4 SmD1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 10.327998 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #14: protein, SmD2

ProteinName: SmD2 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 11.021953 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #15: protein, SmD3

ProteinName: SmD3 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 9.011384 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #16: protein, SmE

ProteinName: SmE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 8.320758 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #17: protein, SmF

ProteinName: SmF / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 8.178299 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #18: protein, SmG

ProteinName: SmG / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 7.662906 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #19: protein, Snu66

ProteinName: Snu66 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 24.367936 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #20: protein, U5 SmE

ProteinName: U5 SmE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 8.362859 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #21: protein, U5 SmD1

ProteinName: U5 SmD1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.200678 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #22: nucleic-acid, U5 snRNA

nucleic acidName: U5 snRNAU5 spliceosomal RNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence: AAGCAGCUUU ACAGAUCAAU GGCGGAGGGA GGUCAACAUC AAGAACUGUG GGCCUUUUAU UGCCUAUAGA ACUUAUAACG AACAUGGUUC UUGCCUUUUA CCAGAACCAU CCGGGUGUUG UCUCCAUAGA AACAGGUAAA GCUGUCCGUU ACUGUGGGCU UGCCAUAUUU ...Sequence:
AAGCAGCUUU ACAGAUCAAU GGCGGAGGGA GGUCAACAUC AAGAACUGUG GGCCUUUUAU UGCCUAUAGA ACUUAUAACG AACAUGGUUC UUGCCUUUUA CCAGAACCAU CCGGGUGUUG UCUCCAUAGA AACAGGUAAA GCUGUCCGUU ACUGUGGGCU UGCCAUAUUU UUUGGAACUU UUCUGCCCUU UUUCUCAAUG AGUAAGGAGG GCGU
MassTheoretical: 45.305805 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #23: protein, Snu13

ProteinName: Snu13NHP2L1 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 13.364584 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #24: protein, LSm2

ProteinName: LSm2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 10.530101 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #25: protein, LSm3

ProteinName: LSm3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 8.747967 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #26: protein, LSm4

ProteinName: LSm4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 8.594071 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #27: protein, LSm5

ProteinName: LSm5 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 8.458736 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #28: protein, LSm6

ProteinName: LSm6 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 8.227233 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #29: protein, LSm7

ProteinName: LSm7 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 7.233692 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #30: protein, LSm8

ProteinName: LSm8 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 7.1935 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #31: protein, Snu114

ProteinName: Snu114 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 97.172828 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.2 mg/mL / pH: 7.9
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 38 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 81000.0 X (nominal), 35714.0 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 500.0 - 3500.0 nm / Energy filter: GIF Quantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 36824
3D reconstructionSoftware: RELION / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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