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- EMDB-2966: EM structure of the yeast spliceosomal U4/U6.U5 tri-snRNP -

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Basic information

Entry
Database: EMDB / ID: EMD-2966
TitleEM structure of the yeast spliceosomal U4/U6.U5 tri-snRNP
Map dataReconstruction of the yeast U4/U6.U5 tri-snRNP
Sample
  • Sample: Yeast U4/U6.U5 tri-snRNP
  • RNA: U4 snRNA
  • RNA: U5 snRNA
  • RNA: U6 snRNA
  • Protein or peptide: U4/U6.U5 tri-snRNP
KeywordsSpliceosome / tri-snRNP
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsNguyen THD / Galej WP / Bai X-C / Savva CG / Newman AJ / Scheres SHW / Nagai K
CitationJournal: 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.
History
DepositionMar 29, 2015-
Header (metadata) releaseMay 6, 2015-
Map releaseJul 1, 2015-
UpdateJul 1, 2015-
Current statusJul 1, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2966.png

Downloads & links

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Map

FileDownload / File: emd_2966.map.gz / Format: CCP4 / Size: 81.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the yeast U4/U6.U5 tri-snRNP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.77 Å/pix.
x 280 pix.
= 495.6 Å		1.77 Å/pix.
x 280 pix.
= 495.6 Å		1.77 Å/pix.
x 280 pix.
= 495.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.77 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.19736847 - 0.37212843
Average (Standard dev.)0.00018776 (±0.01347888)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 495.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.771.771.77
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z495.600495.600495.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.1970.3720.000

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Supplemental data

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Sample components

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Entire : Yeast U4/U6.U5 tri-snRNP

EntireName: Yeast U4/U6.U5 tri-snRNP
Components
  • Sample: Yeast U4/U6.U5 tri-snRNP
  • RNA: U4 snRNA
  • RNA: U5 snRNA
  • RNA: U6 snRNA
  • Protein or peptide: U4/U6.U5 tri-snRNP

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Supramolecule #1000: Yeast U4/U6.U5 tri-snRNP

SupramoleculeName: Yeast U4/U6.U5 tri-snRNP / type: sample / ID: 1000 / Number unique components: 4
Molecular weightTheoretical: 1.5 MDa

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Macromolecule #1: U4 snRNA

MacromoleculeName: U4 snRNA / type: rna / ID: 1 / Classification: OTHER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: budding yeast

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Macromolecule #2: U5 snRNA

MacromoleculeName: U5 snRNA / type: rna / ID: 2 / Classification: OTHER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: budding yeast

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Macromolecule #3: U6 snRNA

MacromoleculeName: U6 snRNA / type: rna / ID: 3 / Classification: OTHER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: budding yeast

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Macromolecule #4: U4/U6.U5 tri-snRNP

MacromoleculeName: U4/U6.U5 tri-snRNP / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Budding yeast / Organelle: Nucleus

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.9
GridDetails: 400 mesh copper grid with thin carbon support, glow discharged in amylamine atmosphere
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 85 K / Instrument: FEI VITROBOT MARK II / Method: Blot for 2 s before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 80 K / Max: 90 K / Average: 85 K
DateJun 10, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 2035 / Average electron dose: 40 e/Å2
Details: Every image is the average of sixteen frames recorded by the direct electron detector
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 79096 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal magnification: 59000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.9 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION
Details: To correct for beam-induced movements, the 16 video frames for each micrograph were first aligned using whole-image motion correction. Second, particle based beam-induced movement correction ...Details: To correct for beam-induced movements, the 16 video frames for each micrograph were first aligned using whole-image motion correction. Second, particle based beam-induced movement correction was performed using statistical movie processing in RELION.
Number images used: 179079
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4i43

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

4bgd

SoftwareName: Chimera, coot
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 3

Initial modelPDB ID:

4wzj

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 4

Initial modelPDB ID:

1qgv

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 5

Initial modelPDB ID:

2ozb

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 6

Initial modelPDB ID:

2ale

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 7

Initial modelPDB ID:

4m77

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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