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- EMDB-6562: The cryo-EM structure of yeast spliceosomal U4/U6.U5 tri-snRNP (i... -

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Entry
Database: EMDB / ID: EMD-6562
TitleThe cryo-EM structure of yeast spliceosomal U4/U6.U5 tri-snRNP (improved map for U4/U6 and U5 RNA region at 3.75 A)
Map data
SampleU4/U6.U5 tri-snRNP from Saccharomyces cerevisiae:
U4/U6.U5 tri-snRNP
Keywordstri-snRNP / pre-mRNA splicing
Function / homology
Function and homology information


spliceosomal conformational changes to generate catalytic conformation / U4/U6 snRNP / snoRNA splicing / Lsm1-7-Pat1 complex / U6 snRNP / rRNA 2'-O-methylation / positive regulation of RNA binding / generation of catalytic spliceosome for first transesterification step / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / splicing factor binding ...spliceosomal conformational changes to generate catalytic conformation / U4/U6 snRNP / snoRNA splicing / Lsm1-7-Pat1 complex / U6 snRNP / rRNA 2'-O-methylation / positive regulation of RNA binding / generation of catalytic spliceosome for first transesterification step / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / splicing factor binding / box C/D RNP complex / P-body assembly / pICln-Sm protein complex / U4 snRNP / nuclear-transcribed mRNA catabolic process / sno(s)RNA-containing ribonucleoprotein complex / small nuclear ribonucleoprotein complex / spliceosomal tri-snRNP complex / SMN-Sm protein complex / U4 snRNA binding / U2-type catalytic step 2 spliceosome / commitment complex / U2 snRNP / U3 snoRNA binding / 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 / RNA metabolic process / tRNA processing / spliceosomal tri-snRNP complex assembly / rRNA methylation / 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 / maturation of SSU-rRNA / U1 snRNA binding / small-subunit processome / isopeptidase activity / catalytic step 2 spliceosome / maturation of LSU-rRNA / spliceosomal complex / P-body / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / metallopeptidase activity / rRNA processing / mRNA splicing, via spliceosome / ribonucleoprotein complex / RNA helicase / RNA helicase activity / GTPase activity / mRNA binding / GTP binding / nucleolus / mitochondrion / RNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
PRP1 splicing factor, N-terminal / Domain of unknown function DUF1115 / Immunoglobulin E-set / Ribosomal protein S5 domain 2-type fold, subgroup / Helicase superfamily 1/2, ATP-binding domain / Pre-mRNA-splicing factor 3 / Tetratricopeptide repeat-containing domain / PROCT domain / NOSIC / PROCN domain ...PRP1 splicing factor, N-terminal / Domain of unknown function DUF1115 / Immunoglobulin E-set / Ribosomal protein S5 domain 2-type fold, subgroup / Helicase superfamily 1/2, ATP-binding domain / Pre-mRNA-splicing factor 3 / Tetratricopeptide repeat-containing domain / PROCT domain / NOSIC / PROCN domain / PRO8NT domain / Ribonuclease H-like superfamily / Tetratricopeptide-like helical domain superfamily / DEAD/DEAH box helicase domain / LSM domain superfamily / Translation protein, beta-barrel domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Translation elongation factor EFG/EF2, domain IV / Sec63 domain / Translation elongation factor EFTu-like, domain 2 / Dim1 family / Ribosomal protein L7Ae/L30e/S12e/Gadd45 / Ribosomal protein L7Ae conserved site / HAT (Half-A-TPR) repeat / Nop domain / H/ACA ribonucleoprotein complex, subunit Nhp2, eukaryote / WD40 repeat / Helicase, C-terminal / LSM domain, eukaryotic/archaea-type / Translational (tr)-type GTP-binding domain / Elongation factor EFG, domain V-like / Pre-mRNA processing factor 4 (PRP4)-like / Sm-like protein Lsm6/SmF / 116kDa U5 small nuclear ribonucleoprotein component, C-terminal / Pre-mRNA-processing factor 6/Prp1/STA1 / Sm-like protein Lsm3 / Sm-like protein Lsm8 / Sm-like protein Lsm4 / Small nuclear ribonucleoprotein F / Small nuclear ribonucleoprotein Sm D3 / Small nuclear ribonucleoprotein G / Sm-like protein LSm5 / 116kDa U5 small nuclear ribonucleoprotein component, N-terminal / 50S ribosomal protein L30e-like / Pre-mRNA-processing-splicing factor 8 / P-loop containing nucleoside triphosphate hydrolase / Small nuclear ribonucleoprotein Sm D2 / Like-Sm (LSM) domain containing protein, LSm4/SmD1/SmD3 / U4/U6 small nuclear ribonucleoprotein Prp31 / U6 snRNA-associated Sm-like protein LSm2 / U4/U6 small nuclear ribonucleoprotein Prp3 / Small nuclear ribonucleoprotein E / PRP8 domain IV core / Ribosomal protein S5 domain 2-type fold / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Tetratricopeptide repeat / RNA recognition motif, spliceosomal PrP8 / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding / Pre-mRNA-processing-splicing factor 8, U6-snRNA-binding / Prp31 C-terminal / Ribosomal protein L7Ae/L8/Nhp2 family / WD40-repeat-containing domain / Sm-like protein Lsm7 / JAB1/MPN/MOV34 metalloenzyme domain / EF-G domain III/V-like / Nop domain superfamily / Snu114, GTP-binding domain / Prp8 RNase domain IV, fingers region / Prp8 RNase domain IV, palm region / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding domain superfamily / Nop, C-terminal domain / Brr2, N-terminal helicase PWI domain / U6 snRNA-associated Sm-like protein Lsm3 / MPN domain / C2 domain superfamily / Winged helix DNA-binding domain superfamily / WD40-repeat-containing domain superfamily / Thioredoxin-like superfamily
U6 snRNA-associated Sm-like protein LSm8 / Spliceosomal protein DIB1 / U6 snRNA-associated Sm-like protein LSm6 / Small nuclear ribonucleoprotein Sm D2 / U4/U6 small nuclear ribonucleoprotein PRP3 / Small nuclear ribonucleoprotein Sm D1 / U6 snRNA-associated Sm-like protein LSm3 / Small nuclear ribonucleoprotein F / U6 snRNA-associated Sm-like protein LSm7 / Pre-mRNA-processing factor 31 ...U6 snRNA-associated Sm-like protein LSm8 / Spliceosomal protein DIB1 / U6 snRNA-associated Sm-like protein LSm6 / Small nuclear ribonucleoprotein Sm D2 / U4/U6 small nuclear ribonucleoprotein PRP3 / Small nuclear ribonucleoprotein Sm D1 / U6 snRNA-associated Sm-like protein LSm3 / Small nuclear ribonucleoprotein F / U6 snRNA-associated Sm-like protein LSm7 / Pre-mRNA-processing factor 31 / U6 snRNA-associated Sm-like protein LSm2 / Small nuclear ribonucleoprotein Sm D3 / Small nuclear ribonucleoprotein G / U6 snRNA-associated Sm-like protein LSm5 / U6 snRNA-associated Sm-like protein LSm4 / Small nuclear ribonucleoprotein-associated protein B / 13 kDa ribonucleoprotein-associated protein / Pre-mRNA-splicing factor SNU114 / Pre-mRNA-splicing factor 8 / Pre-mRNA-splicing helicase BRR2 / U4/U6 small nuclear ribonucleoprotein PRP4 / Pre-mRNA-splicing factor 6 / Small nuclear ribonucleoprotein E
Biological speciesSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsWan R / Yan C / Bai R / Wang L / Huang M / Wong C / Shi Y
CitationJournal: Science / Year: 2016
Title: The 3.8 Å structure of the U4/U6.U5 tri-snRNP: Insights into spliceosome assembly and catalysis.
Authors: Ruixue Wan / Chuangye Yan / Rui Bai / Lin Wang / Min Huang / Catherine C L Wong / Yigong Shi /
Abstract: Splicing of precursor messenger RNA is accomplished by a dynamic megacomplex known as the spliceosome. Assembly of a functional spliceosome requires a preassembled U4/U6.U5 tri-snRNP complex, which ...Splicing of precursor messenger RNA is accomplished by a dynamic megacomplex known as the spliceosome. Assembly of a functional spliceosome requires a preassembled U4/U6.U5 tri-snRNP complex, which comprises the U5 small nuclear ribonucleoprotein (snRNP), the U4 and U6 small nuclear RNA (snRNA) duplex, and a number of protein factors. Here we report the three-dimensional structure of a Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at an overall resolution of 3.8 angstroms by single-particle electron cryomicroscopy. The local resolution for the core regions of the tri-snRNP reaches 3.0 to 3.5 angstroms, allowing construction of a refined atomic model. Our structure contains U5 snRNA, the extensively base-paired U4/U6 snRNA, and 30 proteins including Prp8 and Snu114, which amount to 8495 amino acids and 263 nucleotides with a combined molecular mass of ~1 megadalton. The catalytic nucleotide U80 from U6 snRNA exists in an inactive conformation, stabilized by its base-pairing interactions with U4 snRNA and protected by Prp3. Pre-messenger RNA is bound in the tri-snRNP through base-pairing interactions with U6 snRNA and loop I of U5 snRNA. This structure, together with that of the spliceosome, reveals the molecular choreography of the snRNAs in the activation process of the spliceosomal ribozyme.
History
DepositionDec 23, 2015-
Header (metadata) releaseFeb 10, 2016-
Map releaseFeb 10, 2016-
UpdateFeb 10, 2016-
Current statusFeb 10, 2016Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0135
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0135
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jcm
  • Surface level: 0.0135
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Structure viewerEM map:
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Downloads & links

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Map

FileDownload / File: emd_6562.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 320 pix.
= 422.4 Å
1.32 Å/pix.
x 320 pix.
= 422.4 Å
1.32 Å/pix.
x 320 pix.
= 422.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.0135 / Movie #1: 0.0135
Minimum - Maximum-0.11463609 - 0.19528407
Average (Standard dev.)-0.00003407 (±0.0053946)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z422.400422.400422.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1150.195-0.000

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Supplemental data

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Sample components

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Entire U4/U6.U5 tri-snRNP from Saccharomyces cerevisiae

EntireName: U4/U6.U5 tri-snRNP from Saccharomyces cerevisiae / Number of components: 34

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Component #1: cellular-component, U4/U6.U5 tri-snRNP

Cellular-componentName: U4/U6.U5 tri-snRNP / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

ImagingMicroscope: FEI TITAN / Date: Aug 8, 2015
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
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: 3141 / Scanner: TEMSCAN

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 172134
3D reconstructionSoftware: RELION / Resolution: 3.75 Å / Resolution method: FSC 0.143, gold-standard

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Atomic model buiding

Output model

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