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- EMDB-6565: The cryo-EM structure of yeast spliceosomal U4/U6.U5 tri-snRNP (i... -

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Entry
Database: EMDB / ID: 6565
TitleThe cryo-EM structure of yeast spliceosomal U4/U6.U5 tri-snRNP (improved map for Prp8 region at 3.49 A)
Map dataReconstruction of tri-snRNP
SampleU4/U6.U5 tri-snRNP from Saccharomyces cerevisiae:
U4/U6.U5 tri-snRNP
Keywordstri-snRNP / pre-mRNA splicing
Function / homologyU6 snRNA-associated Sm-like protein LSm2 / U4/U6 small nuclear ribonucleoprotein Prp3 / RNA recognition motif, spliceosomal PrP8 / Tetratricopeptide repeat / WD40 repeat, conserved site / G-protein beta WD-40 repeat / Ribosomal protein S5 domain 2-type fold / PRP8 domain IV core / Small nuclear ribonucleoprotein E / U4/U6 small nuclear ribonucleoprotein Prp31 ...U6 snRNA-associated Sm-like protein LSm2 / U4/U6 small nuclear ribonucleoprotein Prp3 / RNA recognition motif, spliceosomal PrP8 / Tetratricopeptide repeat / WD40 repeat, conserved site / G-protein beta WD-40 repeat / Ribosomal protein S5 domain 2-type fold / PRP8 domain IV core / Small nuclear ribonucleoprotein E / U4/U6 small nuclear ribonucleoprotein Prp31 / Pre-mRNA-processing-splicing factor 8, U6-snRNA-binding / U4/U6 small nuclear ribonucleoprotein Prp4 / Pre-mRNA-processing factor 6/Prp1/STA1 / 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 / 50S ribosomal protein L30e-like / 116kDa U5 small nuclear ribonucleoprotein component, N-terminal / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding / Prp31 C-terminal / Small nuclear ribonucleoprotein G / Tetratricopeptide repeat-containing domain / DEAD/DEAH box helicase domain / Tetratricopeptide-like helical domain superfamily / Ribonuclease H-like superfamily / PRO8NT domain / PROCN domain / NOSIC / PROCT domain / Pre-mRNA-splicing factor 3 / Ribosomal protein L7Ae/L8/Nhp2 family / Helicase superfamily 1/2, ATP-binding domain / Ribosomal protein S5 domain 2-type fold, subgroup / Immunoglobulin E-set / Pre-mRNA processing factor 4 (PRP4)-like / WD40/YVTN repeat-like-containing domain superfamily / Sm-like protein Lsm6/SmF / Sm-like protein Lsm7 / WD40-repeat-containing domain / Sm-like protein LSm5 / Small nuclear ribonucleoprotein Sm D3 / Domain of unknown function DUF1115 / PROCN (NUC071) domain / Sec63 Brl domain / Mitosis protein DIM1 / Elongation factor Tu domain 2 / Elongation factor G, domain IV / PRP1 splicing factor, N-terminal / Protein of unknown function (DUF1115) / PRO8NT (NUC069), PrP8 N-terminal domain / PROCT (NUC072) domain / LSM domain / pre-mRNA processing factor 3 (PRP3) / Prp31 C terminal domain / U6-snRNA interacting domain of PrP8 / U5-snRNA binding site 2 of PrP8 / RNA recognition motif of the spliceosomal PrP8 / PRP8 domain IV core / 116 kDa U5 small nuclear ribonucleoprotein component N-terminus / Trp-Asp (WD) repeats signature. / snoRNA binding domain, fibrillarin / Ribosomal protein L7Ae/L30e/S12e/Gadd45 family / Small nuclear ribonucleoprotein F / Nop domain superfamily / Sm-like protein Lsm4 / Small nuclear ribonucleoprotein D1 / Sm-like protein Lsm8 / Sm-like protein Lsm3 / EF-G domain III/V-like / 116kDa U5 small nuclear ribonucleoprotein component, C-terminal / C2 domain superfamily / Thioredoxin-like superfamily / Elongation factor G C-terminus / WD40-repeat-containing domain superfamily / Winged helix DNA-binding domain superfamily / MPN domain / U6 snRNA-associated Sm-like protein Lsm3 / Elongation factor Tu GTP binding domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / WD domain, G-beta repeat / LSM domain superfamily / PRP1 splicing factor, N-terminal / Trp-Asp (WD) repeats profile. / JAB1/MPN/MOV34 metalloenzyme domain / mRNA Splicing - Minor Pathway / Major pathway of rRNA processing in the nucleolus and cytosol / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / Formation of TC-NER Pre-Incision Complex / mRNA decay by 5' to 3' exoribonuclease / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Nop domain profile. / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / Trp-Asp (WD) repeats circular profile. / Translation protein, beta-barrel domain superfamily / MPN domain profile. / Ribosomal protein L7Ae signature.
Function and homology information
SourceSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / 3.49 Å resolution
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.
Validation ReportPDB-ID: 3jcm

SummaryFull reportAbout validation report
DateDeposition: Dec 23, 2015 / Header (metadata) release: Feb 10, 2016 / Map release: Feb 10, 2016 / Last update: Feb 10, 2016

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0222
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0222
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-3jcm
  • Surface level: 0.0222
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_6565.map.gz (map file in CCP4 format, 128001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
320 pix
1.32 Å/pix.
= 422.4 Å
320 pix
1.32 Å/pix.
= 422.4 Å
320 pix
1.32 Å/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 Level:0.0222 (by author), 0.0222 (movie #1):
Minimum - Maximum-0.16161779 - 0.28660139
Average (Standard dev.)-0.00005935 (0.00496697)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions320320320
Origin000
Limit319319319
Spacing320320320
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 deg.

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
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1620.287-0.000

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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

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / 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: 3.49 Å / Resolution method: FSC 0.143, gold-standard

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Atomic model buiding

Output model

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