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- EMDB-8013: Head region of the yeast spliceosomal U4/U6.U5 tri-snRNP -

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Basic information

Entry
Database: EMDB / ID: EMD-8013
TitleHead region of the yeast spliceosomal U4/U6.U5 tri-snRNP
Map data
SampleHead region of the yeast spliceosomal U4/U6.U5 tri-snRNP
  • Small nuclear ribonucleoprotein-associated protein B
  • (Small nuclear ribonucleoprotein ...SnRNP) x 6
  • Snu66
  • (Pre-mRNA-splicing ...) x 2
  • nucleic-acidNucleic acid
Function / homology
Function and homology information


maturation of 5S rRNA / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / splicing factor binding / pICln-Sm protein complex / U4 snRNP / small nuclear ribonucleoprotein complex / spliceosomal tri-snRNP complex / SMN-Sm protein complex / mRNA cis splicing, via spliceosome / commitment complex ...maturation of 5S rRNA / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / splicing factor binding / pICln-Sm protein complex / U4 snRNP / small nuclear ribonucleoprotein complex / spliceosomal tri-snRNP complex / SMN-Sm protein complex / mRNA cis splicing, via spliceosome / commitment complex / U2 snRNP / U12-type spliceosomal complex / generation of catalytic spliceosome for second transesterification step / U2-type prespliceosome / spliceosomal snRNP assembly / U1 snRNP / mRNA 3'-splice site recognition / mRNA 5'-splice site recognition / precatalytic spliceosome / spliceosomal tri-snRNP complex assembly / U5 snRNP / U5 snRNA binding / poly(U) RNA binding / U2 snRNA binding / U6 snRNA binding / pre-mRNA intronic binding / U4/U6 x U5 tri-snRNP complex / U1 snRNA binding / isopeptidase activity / catalytic step 2 spliceosome / spliceosomal complex / metallopeptidase activity / mRNA splicing, via spliceosome / ribonucleoprotein complex / RNA helicase / RNA helicase activity / mRNA binding / RNA binding / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
JAB1/MPN/MOV34 metalloenzyme domain / Helicase superfamily 1/2, ATP-binding domain / LSM domain, eukaryotic/archaea-type / Helicase, C-terminal / Sec63 domain / SNU66/SART1 family / LSM domain superfamily / DEAD/DEAH box helicase domain / Ribonuclease H-like superfamily / PRO8NT domain ...JAB1/MPN/MOV34 metalloenzyme domain / Helicase superfamily 1/2, ATP-binding domain / LSM domain, eukaryotic/archaea-type / Helicase, C-terminal / Sec63 domain / SNU66/SART1 family / LSM domain superfamily / DEAD/DEAH box helicase domain / Ribonuclease H-like superfamily / PRO8NT domain / PROCN domain / PROCT domain / Immunoglobulin E-set / Small nuclear ribonucleoprotein F / Sm-like protein Lsm6/SmF / Pre-mRNA-processing-splicing factor 8, U6-snRNA-binding / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding / RNA recognition motif, spliceosomal PrP8 / PRP8 domain IV core / Small nuclear ribonucleoprotein E / Like-Sm (LSM) domain containing protein, LSm4/SmD1/SmD3 / Small nuclear ribonucleoprotein Sm D2 / P-loop containing nucleoside triphosphate hydrolase / Pre-mRNA-processing-splicing factor 8 / Small nuclear ribonucleoprotein Sm D3 / Small nuclear ribonucleoprotein G / Winged helix DNA-binding domain superfamily / Prp8 RNase domain IV, fingers region / Prp8 RNase domain IV, palm region / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding domain superfamily / Brr2, N-terminal helicase PWI domain / MPN domain / C2 domain superfamily
Small nuclear ribonucleoprotein F / Small nuclear ribonucleoprotein E / Small nuclear ribonucleoprotein Sm D2 / Small nuclear ribonucleoprotein Sm D1 / Pre-mRNA-splicing factor 8 / Small nuclear ribonucleoprotein Sm D3 / Small nuclear ribonucleoprotein G / Small nuclear ribonucleoprotein-associated protein B / Pre-mRNA-splicing helicase BRR2 / 66 kDa U4/U6.U5 small nuclear ribonucleoprotein component
Biological speciesSaccharomyces cerevisiae (baker's yeast) / Baker's yeast (baker's yeast)
Methodsingle particle reconstruction / Resolution: 4.2 Å
AuthorsNguyen THD / Galej WP / Bai XC / Oubridge C / Scheres SHW / Newman AJ / Nagai K
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom) United Kingdom
Citation
Journal: 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.
#1: Journal: Nature / Year: 2015
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.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionDec 15, 2015-
Header (metadata) releaseJan 27, 2016-
Map releaseJan 27, 2016-
UpdateOct 2, 2019-
Current statusOct 2, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
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  • Surface view with fitted model
  • Atomic models: PDB-5gao
  • Surface level: 0.02
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8013.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: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.07287737 - 0.15714578
Average (Standard dev.)-0.00023307164 (±0.003403494)
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-0.0730.157-0.000

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Supplemental data

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Sample components

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Entire Head region of the yeast spliceosomal U4/U6.U5 tri-snRNP

EntireName: Head region of the yeast spliceosomal U4/U6.U5 tri-snRNP
Number of components: 12

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Component #1: protein, Head region of the yeast spliceosomal U4/U6.U5 tri-snRNP

ProteinName: Head region of the yeast spliceosomal U4/U6.U5 tri-snRNP
Recombinant expression: No
MassTheoretical: 400 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123

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Component #2: protein, Small nuclear ribonucleoprotein-associated protein B

ProteinName: Small nuclear ribonucleoprotein-associated protein B
Details: The SmB protein from the U4 snRNP Sm protein ring. The C-terminus is disordered in this and other previously reported structures.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 22.42699 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BCY123

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Component #3: protein, Small nuclear ribonucleoprotein Sm D1

ProteinName: Small nuclear ribonucleoprotein Sm D1
Details: The SmD1 protein from the U4 snRNP Sm protein ring.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 16.296798 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BCY123

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Component #4: protein, Small nuclear ribonucleoprotein Sm D2

ProteinName: Small nuclear ribonucleoprotein Sm D2
Details: The SmD2 protein from the U4 snRNP Sm protein ring.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 12.876066 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BCY123

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Component #5: protein, Small nuclear ribonucleoprotein Sm D3

ProteinName: Small nuclear ribonucleoprotein Sm D3
Details: The SmD3 protein from the U4 snRNP Sm protein ring.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 11.240139 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BCY123

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Component #6: protein, Small nuclear ribonucleoprotein E

ProteinName: Small nuclear ribonucleoprotein E / Details: The SmE protein from the U4 snRNP Sm protein ring. / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 10.385098 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BCY123

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Component #7: protein, Small nuclear ribonucleoprotein F

ProteinName: Small nuclear ribonucleoprotein F / Details: The SmF protein from the U4 snRNP Sm protein ring. / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.669945 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BCY123

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Component #8: protein, Small nuclear ribonucleoprotein G

ProteinName: Small nuclear ribonucleoprotein G / Details: The SmG protein from the U4 snRNP Sm protein ring. / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 8.490809 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BCY123

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Component #9: protein, Snu66

ProteinName: Snu66
Details: NB: Snu66 protein has been mostly fitted as poly(Ala) into helices and extended polypeptide within the EM map. The authors do not believe the numbering to be accurate and it is likely incorrect.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 25.078721 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BCY123

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Component #10: protein, Pre-mRNA-splicing helicase BRR2

ProteinName: Pre-mRNA-splicing helicase BRR2
Details: Brr2 RNA helicase loaded onto U4 snRNA strand of the U4/U6 duplex between Stem 1 duplex and the 3' stem loop.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 246.470266 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BCY123

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Component #11: nucleic-acid, Saccharomyces cerevisiae strain UOA_M2 chromosome 5...

nucleic acidName: Saccharomyces cerevisiae strain UOA_M2 chromosome 5 sequence
Class: RNA
Details: This U4 snRNA region has a disordered region between stem 1 of the U4/U6 snRNA duplex and where the RNA is loaded into Brr2 protein. The apical part of the 3' stem loop and the region ...Details: This U4 snRNA region has a disordered region between stem 1 of the U4/U6 snRNA duplex and where the RNA is loaded into Brr2 protein. The apical part of the 3' stem loop and the region following the Sm site are also disordered.
Structure: OTHER / Synthetic: No
Sequence:
GAAAUUUAAU UAUAAACCAG ACCGUCUCCU CAUGGUCAAU UCGGUGUUCG CUUUUGAAUA CUUCAAGACU AUGUAGGGAA UUUUUGGAAU ACCUUU
MassTheoretical: 30.615994 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BCY123

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Component #12: protein, Pre-mRNA-splicing factor 8

ProteinName: Pre-mRNA-splicing factor 8
Details: Prp8 Jab1/MPN domain is connected to the rest of the Prp8 protein by a flexible linker peptide.
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 30.231008 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: BCY123

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle
Sample solutionSpecimen conc.: 0.2 mg/mL / pH: 7.9
Support filmGrids are made of holey carbon, carbon-coated and glow discharged in N-amylamine.
VitrificationCryogen name: NONE

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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 acquisition

Image acquisitionNumber of digital images: 2477

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 140155
3D reconstructionSoftware: RELION / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: RECIPROCAL
Input PDB model: 4BGD, 4BGD
Chain ID: B, A
Output model

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