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- EMDB-4532: Human post-catalytic spliceosome (P complex), focused refinement ... -

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Basic information

Entry
Database: EMDB / ID: EMD-4532
TitleHuman post-catalytic spliceosome (P complex), focused refinement on DHX8
Map dataHuman post-catalytic spliceosome (P complex), focused refinement on DHX8
Sample
  • Complex: Human post-catalytic P complex spliceosome
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 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.
History
DepositionJan 3, 2019-
Header (metadata) releaseMay 15, 2019-
Map releaseMay 15, 2019-
UpdateMay 15, 2019-
Current statusMay 15, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4532.png

Downloads & links

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Map

FileDownload / File: emd_4532.map.gz / Format: CCP4 / Size: 262.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman post-catalytic spliceosome (P complex), focused refinement on DHX8
Voxel sizeX=Y=Z: 1.2 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.08708665 - 0.1541328
Average (Standard dev.)-0.000030058103 (±0.002890318)
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-0.0870.154-0.000

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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
Components
  • Complex: Human post-catalytic P complex spliceosome

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Supramolecule #1: Human post-catalytic P complex spliceosome

SupramoleculeName: Human post-catalytic P complex spliceosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#44
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 3.0 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 mg/mL
BufferpH: 7.9
Component:
ConcentrationNameFormula
20.0 mMHEPES
100.0 mMKCl
0.2 mMEDTAEthylenediaminetetraacetic acid
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 25s, then blotted for 2.5-3.5s and immediately plunged into liquid ethane..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 156809
FSC plot (resolution estimation)

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