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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: EMD-3980
TitlePost-catalytic P complex spliceosome with 3' splice site undocked
Map dataNone
Sample
  • Complex: P complex spliceosome with 3' splice site undocked
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
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.
History
DepositionNov 8, 2017-
Header (metadata) releaseDec 6, 2017-
Map releaseDec 6, 2017-
UpdateDec 20, 2017-
Current statusDec 20, 2017Processing site: PDBe / Status: Released

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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
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3980.png

Downloads & links

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Map

FileDownload / File: emd_3980.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.12 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.035
Minimum - Maximum-0.078982145 - 0.14612406
Average (Standard dev.)-0.00014100836 (±0.006374927)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 537.6 Å
α=β=γ: 90.0 °

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
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
Components
  • Complex: P complex spliceosome with 3' splice site undocked

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Supramolecule #1: P complex spliceosome with 3' splice site undocked

SupramoleculeName: P complex spliceosome with 3' splice site undocked / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#30
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.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 2.2 MDa

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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

Concentration1.4 mg/mL
BufferpH: 7.9
Component:
ConcentrationNameFormula
20.0 millimolarHepes.KOH pH 7.9
75.0 millimolarpotassium chlorideKCl
250.0 micromolarEDTAEthylenediaminetetraacetic acid
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 7.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.2 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-20 / Number grids imaged: 1 / Number real images: 2295 / Average exposure time: 12.0 sec. / Average electron dose: 47.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 206529
CTF correctionSoftware - Name: Gctf
Startup modelType of model: EMDB MAP
EMDB ID:

3539

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final 3D classificationSoftware - Name: RELION (ver. 2.1) / Software - details: patched
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1) / Software - details: patched
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Software - details: patched / Number images used: 24395
FSC plot (resolution estimation)

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