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- EMDB-4365: Prespliceosome structure provides insight into spliceosome assemb... -

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Basic information

Entry
Database: EMDB / ID: EMD-4365
TitlePrespliceosome structure provides insight into spliceosome assembly and regulation (map A3)
Map dataUnsharpened A3 map
Sample
  • Complex: Prespliceosome A complex
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.4 Å
AuthorsPlaschka C / Lin P-C / Charenton C / Nagai K
CitationJournal: Nature / Year: 2018
Title: Prespliceosome structure provides insights into spliceosome assembly and regulation.
Authors: Clemens Plaschka / Pei-Chun Lin / Clément Charenton / Kiyoshi Nagai /
Abstract: The spliceosome catalyses the excision of introns from pre-mRNA in two steps, branching and exon ligation, and is assembled from five small nuclear ribonucleoprotein particles (snRNPs; U1, U2, U4, ...The spliceosome catalyses the excision of introns from pre-mRNA in two steps, branching and exon ligation, and is assembled from five small nuclear ribonucleoprotein particles (snRNPs; U1, U2, U4, U5, U6) and numerous non-snRNP factors. For branching, the intron 5' splice site and the branch point sequence are selected and brought by the U1 and U2 snRNPs into the prespliceosome, which is a focal point for regulation by alternative splicing factors. The U4/U6.U5 tri-snRNP subsequently joins the prespliceosome to form the complete pre-catalytic spliceosome. Recent studies have revealed the structural basis of the branching and exon-ligation reactions, however, the structural basis of the early events in spliceosome assembly remains poorly understood. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae prespliceosome at near-atomic resolution. The structure reveals an induced stabilization of the 5' splice site in the U1 snRNP, and provides structural insights into the functions of the human alternative splicing factors LUC7-like (yeast Luc7) and TIA-1 (yeast Nam8), both of which have been linked to human disease. In the prespliceosome, the U1 snRNP associates with the U2 snRNP through a stable contact with the U2 3' domain and a transient yeast-specific contact with the U2 SF3b-containing 5' region, leaving its tri-snRNP-binding interface fully exposed. The results suggest mechanisms for 5' splice site transfer to the U6 ACAGAGA region within the assembled spliceosome and for its subsequent conversion to the activation-competent B-complex spliceosome. Taken together, the data provide a working model to investigate the early steps of spliceosome assembly.
History
DepositionApr 10, 2018-
Header (metadata) releaseMay 16, 2018-
Map releaseJul 18, 2018-
UpdateAug 1, 2018-
Current statusAug 1, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0149
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0149
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6g90
  • Surface level: 0.0149
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4365.map.gz / Format: CCP4 / Size: 669.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened A3 map
Voxel sizeX=Y=Z: 1.13 Å
Density
Contour LevelBy AUTHOR: 0.0149 / Movie #1: 0.0149
Minimum - Maximum-0.012716197 - 0.050490625
Average (Standard dev.)0.00026135272 (±0.0021952535)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions560560560
Spacing560560560
CellA=B=C: 632.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.131.131.13
M x/y/z560560560
origin x/y/z0.0000.0000.000
length x/y/z632.800632.800632.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS560560560
D min/max/mean-0.0130.0500.000

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Supplemental data

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Sample components

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Entire : Prespliceosome A complex

EntireName: Prespliceosome A complex
Components
  • Complex: Prespliceosome A complex

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Supramolecule #1: Prespliceosome A complex

SupramoleculeName: Prespliceosome A complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#37
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightExperimental: 1.8 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

Concentration0.4 mg/mL
BufferpH: 7.9
Component:
ConcentrationFormulaName
20.0 mMHEPESHEPES pH 7.9
50.0 mMKCl
1.0 %Glycerol
2.0 mMMgCl2
0.001 %NP-40
1.0 mMDTT
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III
Details: Grids were blotted for 2-3.5 s and vitrified by plunging into liquid ethane with a FEI Vitrobot Mark III operated at 4 degree Celsius and 100% humidity..

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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 magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 9407 / Average electron dose: 43.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER
Details: The initial model was generated from 22319 particles using cryoSPARC.
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 19937

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Atomic model buiding 1

RefinementSpace: REAL

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