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- EMDB-4533: Human post-catalytic spliceosome (P complex), focused refinement ... -

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Basic information

Entry
Database: EMDB / ID: EMD-4533
TitleHuman post-catalytic spliceosome (P complex), focused refinement on Prp19
Map data
SampleHuman post-catalytic P complex spliceosome
SourceHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsFica SM / Oubridge C / Wilkinson ME / Newman AJ / Nagai KN
CitationJournal: Science / Year: 2019
Title: A human postcatalytic spliceosome structure reveals essential roles of metazoan factors for exon ligation.
Authors: Sebastian M Fica / Chris Oubridge / Max E Wilkinson / Andrew J Newman / Kiyoshi Nagai /
Abstract: During exon ligation, the spliceosome recognizes the 3'-splice site (3'SS) of precursor messenger RNA (pre-mRNA) through non-Watson-Crick pairing with the 5'SS and the branch adenosine, in a ...During exon ligation, the spliceosome recognizes the 3'-splice site (3'SS) of precursor messenger RNA (pre-mRNA) through non-Watson-Crick pairing with the 5'SS and the branch adenosine, in a conformation stabilized by Prp18 and Prp8. Here we present the 3.3-angstrom cryo-electron microscopy structure of a human postcatalytic spliceosome just after exon ligation. The 3'SS docks at the active site through conserved RNA interactions in the absence of Prp18. Unexpectedly, the metazoan-specific FAM32A directly bridges the 5'-exon and intron 3'SS of pre-mRNA and promotes exon ligation, as shown by functional assays. CACTIN, SDE2, and NKAP-factors implicated in alternative splicing-further stabilize the catalytic conformation of the spliceosome during exon ligation. Together these four proteins act as exon ligation factors. Our study reveals how the human spliceosome has co-opted additional proteins to modulate a conserved RNA-based mechanism for 3'SS selection and to potentially fine-tune alternative splicing at the exon ligation stage.
DateDeposition: Jan 3, 2019 / Header (metadata) release: May 15, 2019 / Map release: May 15, 2019 / Update: May 15, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_4533.map.gz / Format: CCP4 / Size: 262.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.2 Å/pix.
x 410 pix.
= 492. Å
1.2 Å/pix.
x 410 pix.
= 492. Å
1.2 Å/pix.
x 410 pix.
= 492. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.039257515 - 0.07526225
Average (Standard dev.)-0.000015612099 (±0.0021744068)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions410410410
Spacing410410410
CellA=B=C: 492.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21.21.2
M x/y/z410410410
origin x/y/z0.0000.0000.000
length x/y/z492.000492.000492.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ208208208
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS410410410
D min/max/mean-0.0390.075-0.000

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Supplemental data

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Sample components

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Entire Human post-catalytic P complex spliceosome

EntireName: Human post-catalytic P complex spliceosome / Number of components: 1

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Component #1: protein, Human post-catalytic P complex spliceosome

ProteinName: Human post-catalytic P complex spliceosome / Recombinant expression: No
MassTheoretical: 3.0 MDa
SourceSpecies: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/mL / pH: 7.9
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %
Details: 3 uL sample was applied to the grid, left for 25s, then blotted for 2.5-3.5s and immediately plunged into liquid ethane..

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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: 60 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500.0 - 3500.0 nm
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 161681
3D reconstructionSoftware: RELION / Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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