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- EMDB-12115: Yeast C complex spliceosome, reconstructed signal-subtracted map ... -

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Basic information

Entry
Database: EMDB / ID: EMD-12115
TitleYeast C complex spliceosome, reconstructed signal-subtracted map after focused classification on Prp19
Map dataYeast C complex spliceosome, reconstructed signal-subtracted map after focused classification on Prp19
Sample
  • Complex: Yeast C complex spliceosome with Prp18/Slu7 bound
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.3 Å
AuthorsWilkinson ME / Fica SM / Galej WP / Nagai K
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)SPLICE3D United Kingdom
Medical Research Council (MRC, United Kingdom) United Kingdom
Citation
Journal: Mol Cell / Year: 2021
Title: Structural basis for conformational equilibrium of the catalytic spliceosome.
Authors: Max E Wilkinson / Sebastian M Fica / Wojciech P Galej / Kiyoshi Nagai /
Abstract: The ATPase Prp16 governs equilibrium between the branching (B/C) and exon ligation (C/P) conformations of the spliceosome. Here, we present the electron cryomicroscopy reconstruction of the ...The ATPase Prp16 governs equilibrium between the branching (B/C) and exon ligation (C/P) conformations of the spliceosome. Here, we present the electron cryomicroscopy reconstruction of the Saccharomyces cerevisiae C-complex spliceosome at 2.8 Å resolution and identify a novel C-complex intermediate (C) that elucidates the molecular basis for this equilibrium. The exon-ligation factors Prp18 and Slu7 bind to C before ATP hydrolysis by Prp16 can destabilize the branching conformation. Biochemical assays suggest that these pre-bound factors prime the C complex for conversion to C by Prp16. A complete model of the Prp19 complex (NTC) reveals how the branching factors Yju2 and Isy1 are recruited by the NTC before branching. Prp16 remodels Yju2 binding after branching, allowing Yju2 to remain tethered to the NTC in the C complex to promote exon ligation. Our results explain how Prp16 action modulates the dynamic binding of step-specific factors to alternatively stabilize the C or C conformation and establish equilibrium of the catalytic spliceosome.
#1: Journal: To Be Published
Title: Structural basis for conformational equilibrium of the catalytic spliceosome
Authors: Wilkinson ME / Fica SM / Galej WP / Nagai K
History
DepositionDec 14, 2020-
Header (metadata) releaseFeb 3, 2021-
Map releaseFeb 3, 2021-
UpdateApr 14, 2021-
Current statusApr 14, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.013
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12115.png

Downloads & links

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Map

FileDownload / File: emd_12115.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast C complex spliceosome, reconstructed signal-subtracted map after focused classification on Prp19
Voxel sizeX=Y=Z: 1.145 Å
Density
Contour LevelBy AUTHOR: 0.013 / Movie #1: 0.013
Minimum - Maximum-0.0062613804 - 0.028833339
Average (Standard dev.)1.9095107e-06 (±0.0013037217)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 343.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1451.1451.145
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z343.500343.500343.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0060.0290.000

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Supplemental data

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Half map: Yeast C complex spliceosome, reconstructed signal-subtracted map after...

Fileemd_12115_half_map_1.map
AnnotationYeast C complex spliceosome, reconstructed signal-subtracted map after focused classification on Prp19, half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Yeast C complex spliceosome, reconstructed signal-subtracted map after...

Fileemd_12115_half_map_2.map
AnnotationYeast C complex spliceosome, reconstructed signal-subtracted map after focused classification on Prp19, half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Yeast C complex spliceosome with Prp18/Slu7 bound

EntireName: Yeast C complex spliceosome with Prp18/Slu7 bound
Components
  • Complex: Yeast C complex spliceosome with Prp18/Slu7 bound

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Supramolecule #1: Yeast C complex spliceosome with Prp18/Slu7 bound

SupramoleculeName: Yeast C complex spliceosome with Prp18/Slu7 bound / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#40
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 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

BufferpH: 7.9
GridModel: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 9 / Number real images: 24115 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 724244
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 49514
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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