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- EMDB-47164: Yeast post-catalytic P complex spliceosome, focussed refinement o... -

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Basic information

Entry
Database: EMDB / ID: EMD-47164
TitleYeast post-catalytic P complex spliceosome, focussed refinement on the Cwc22 N-terminal domain
Map dataP-complex spliceosome focussed-refinement on the Cwc22 N-terminal domain, postprocessed
Sample
  • Complex: Yeast post-catalytic P complex spliceosome
Keywordsspliceosome / snRNA / yeast / ribonucleoprotein complex / SPLICING
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.57 Å
AuthorsWilkinson ME / Hoskins AA
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI)HHMI Helen Hay Whitney Postdoctoral Fellowship United States
CitationJournal: Elife / Year: 2024
Title: Control of 3' splice site selection by the yeast splicing factor Fyv6.
Authors: Katherine A Senn / Karli A Lipinski / Natalie J Zeps / Amory F Griffin / Max E Wilkinson / Aaron A Hoskins /
Abstract: Pre-mRNA splicing is catalyzed in two steps: 5' splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (first and ...Pre-mRNA splicing is catalyzed in two steps: 5' splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (first and second step factors). We recently identified Fyv6 (FAM192A in humans) as a second step factor in ; however, we did not determine how widespread Fyv6's impact is on the transcriptome. To answer this question, we have used RNA sequencing (RNA-seq) to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3' SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of a yeast product complex spliceosome containing Fyv6 at 2.3 Å. The structure reveals that Fyv6 is the only second step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the first step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3' SS.
History
DepositionOct 1, 2024-
Header (metadata) releaseDec 25, 2024-
Map releaseDec 25, 2024-
UpdateJan 1, 2025-
Current statusJan 1, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47164.png

Downloads & links

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Map

FileDownload / File: emd_47164.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationP-complex spliceosome focussed-refinement on the Cwc22 N-terminal domain, postprocessed
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.97 Å/pix.
x 192 pix.
= 185.856 Å		0.97 Å/pix.
x 192 pix.
= 185.856 Å		0.97 Å/pix.
x 192 pix.
= 185.856 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.968 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.08921542 - 0.1370292
Average (Standard dev.)0.00021810361 (±0.0029811468)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 185.856 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_47164_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: P-complex spliceosome focussed-refinement on the Cwc22 N-terminal domain,...

Fileemd_47164_half_map_1.map
AnnotationP-complex spliceosome focussed-refinement on the Cwc22 N-terminal domain, half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: P-complex spliceosome focussed-refinement on the Cwc22 N-terminal domain,...

Fileemd_47164_half_map_2.map
AnnotationP-complex spliceosome focussed-refinement on the Cwc22 N-terminal domain, half-map 1
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 post-catalytic P complex spliceosome

EntireName: Yeast post-catalytic P complex spliceosome
Components
  • Complex: Yeast post-catalytic P complex spliceosome

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Supramolecule #1: Yeast post-catalytic P complex spliceosome

SupramoleculeName: Yeast post-catalytic P complex spliceosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#41
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 2.27 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

Concentration0.1 mg/mL
BufferpH: 7.9
Component:
ConcentrationName
20.0 mMHEPES
75.0 mMpotassium chloride
0.25 mMEDTA
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 2 / Number real images: 51113 / Average exposure time: 0.72 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 1.3 µm / 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

Particle selectionNumber selected: 1820457
Startup modelType of model: EMDB MAP
EMDB ID:

10140

Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5.0) / Software - details: Blush regularization used / Number images used: 201984
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5.0)
Final 3D classificationNumber classes: 4 / Avg.num./class: 102236 / Software - Name: RELION (ver. 5.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsAfter fixing in ISOLDE, the model was refined in phenix.real_space_refine with the following non-default flags: refinement { run = *minimization_global rigid_body local_grid_search morphing simulated_annealing *adp } reference_model { enabled = True use_starting_model_as_reference = True fix_outliers = False strict_rotamer_matching = True } pdb_interpretation { reference_coordinate_restraints { enabled = True exclude_outliers = False top_out = True } automatic_linking { link_metals = True } peptide_link { restrain_rama_outliers = False restrain_rama_allowed = False } ramachandran_plot_restraints { enabled = False } }
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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