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- EMDB-10140: Post-catalytic P complex spliceosome at 3.3 angstrom resolution f... -

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Basic information

Entry
Database: EMDB / ID: EMD-10140
TitlePost-catalytic P complex spliceosome at 3.3 angstrom resolution from merging data sets
Map dataNone
Sample
  • Complex: P complex spliceosome with 3' exon docked
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsWilkinson ME / Nagai K
Funding support United Kingdom, Belgium, 2 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_U105184330 United Kingdom
European Research CouncilAdG-693087-SPLICE3D Belgium
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
DepositionJul 23, 2019-
Header (metadata) releaseJul 31, 2019-
Map releaseJul 31, 2019-
UpdateSep 18, 2019-
Current statusSep 18, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.023
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10140.png

Downloads & links

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Map

FileDownload / File: emd_10140.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.023 / Movie #1: 0.023
Minimum - Maximum-0.06356835 - 0.11250346
Average (Standard dev.)0.000039320665 (±0.0037345937)
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-0.0640.1130.000

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Supplemental data

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Sample components

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Entire : P complex spliceosome with 3' exon docked

EntireName: P complex spliceosome with 3' exon docked
Components
  • Complex: P complex spliceosome with 3' exon docked

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Supramolecule #1: P complex spliceosome with 3' exon docked

SupramoleculeName: P complex spliceosome with 3' exon docked / type: complex / ID: 1 / Parent: 0
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: 7.8
Component:
ConcentrationNameFormula
20.0 mMHEPES
75.0 mMKCl
0.25 mMEDTA
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 uL sample was applied to the grid, left for 30s, then blotted for 3s and immediately plunged into liquid ethane.

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Electron microscopy #1

Microscopy ID1
MicroscopeFEI TITAN KRIOS
Image recordingImage recording ID: 1 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2384 / Average exposure time: 12.0 sec. / Average electron dose: 47.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #1~

Microscopy ID1
MicroscopeFEI TITAN KRIOS
Image recording#0 - Image recording ID: 2 / #0 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #0 - Detector mode: SUPER-RESOLUTION / #0 - Number grids imaged: 1 / #0 - Number real images: 173 / #0 - Average exposure time: 7.0 sec. / #0 - Average electron dose: 45.0 e/Å2 / #1 - Image recording ID: 3 / #1 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #1 - Detector mode: COUNTING / #1 - Digitization - Frames/image: 1-35 / #1 - Number grids imaged: 1 / #1 - Number real images: 1441 / #1 - Average exposure time: 7.0 sec. / #1 - Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Image recording ID1
Particle selectionNumber selected: 286038
Startup modelType of model: EMDB MAP
EMDB ID:

3979

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 52748
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6exn

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