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- EMDB-3980: Post-catalytic P complex spliceosome with 3' splice site undocked -

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Basic information

Entry
Database: EMDB / ID: 3980
TitlePost-catalytic P complex spliceosome with 3' splice site undocked
Map data
SampleP complex spliceosome with 3' splice site undocked:
SourceSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / 4 Å resolution
AuthorsWilkinson ME / Fica SM / Galej WP / Norman CM / Newman AJ / Nagai K
CitationJournal: Science / Year: 2017
Title: Postcatalytic spliceosome structure reveals mechanism of 3'-splice site selection.
Authors: Max E Wilkinson / Sebastian M Fica / Wojciech P Galej / Christine M Norman / Andrew J Newman / Kiyoshi Nagai
Abstract: Introns are removed from eukaryotic messenger RNA precursors by the spliceosome in two transesterification reactions-branching and exon ligation. The mechanism of 3'-splice site recognition during ...Introns are removed from eukaryotic messenger RNA precursors by the spliceosome in two transesterification reactions-branching and exon ligation. The mechanism of 3'-splice site recognition during exon ligation has remained unclear. Here we present the 3.7-angstrom cryo-electron microscopy structure of the yeast P-complex spliceosome immediately after exon ligation. The 3'-splice site AG dinucleotide is recognized through non-Watson-Crick pairing with the 5' splice site and the branch-point adenosine. After the branching reaction, protein factors work together to remodel the spliceosome and stabilize a conformation competent for 3'-splice site docking, thereby promoting exon ligation. The structure accounts for the strict conservation of the GU and AG dinucleotides at the 5' and 3' ends of introns and provides insight into the catalytic mechanism of exon ligation.
DateDeposition: Nov 8, 2017 / Header (metadata) release: Dec 6, 2017 / Map release: Dec 6, 2017 / Last update: Dec 20, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3980.map.gz (map file in CCP4 format, 442369 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
480 pix
1.12 Å/pix.
= 537.6 Å
480 pix
1.12 Å/pix.
= 537.6 Å
480 pix
1.12 Å/pix.
= 537.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.12 Å
Density
Contour Level:0.04 (by author), 0.035 (movie #1):
Minimum - Maximum-0.078982145 - 0.14612406
Average (Standard dev.)-0.00014100836 (0.006374927)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions480480480
Origin000
Limit479479479
Spacing480480480
CellA=B=C: 537.6 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.121.121.12
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z537.600537.600537.600
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.0790.146-0.000

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Supplemental data

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Sample components

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Entire P complex spliceosome with 3' splice site undocked

EntireName: P complex spliceosome with 3' splice site undocked
Details: P complex was assembled in vitro in splicing extract supplemented with dominant negative mutant Prp22 protein. P complex was purified via MS2-MBP fusion protein bound to 3 MS2 hairpin loops on the mRNA.
Number of components: 1
MassTheoretical: 2.2 MDa

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Component #1: protein, P complex spliceosome with 3' splice site undocked

ProteinName: P complex spliceosome with 3' splice site undocked
Details: P complex was assembled in vitro in splicing extract supplemented with dominant negative mutant Prp22 protein. P complex was purified via MS2-MBP fusion protein bound to 3 MS2 hairpin loops on the mRNA.
Recombinant expression: No
MassTheoretical: 2.2 MDa
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.: 1.4 mg/ml / pH: 7.9
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %
Details: 3 microlitres sample were applied to the grid, left for 15 seconds and then blotted for 3.0 seconds before plunging.

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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: 47 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 105000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 200 - 3000 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2295

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 24395
3D reconstructionSoftware: RELION / Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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