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- EMDB-4059: Structure of the yeast spliceosome immediately after branching. C... -

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Basic information

Entry
Database: EMDB / ID: EMD-4059
TitleStructure of the yeast spliceosome immediately after branching. Core with WD40 domain.
Map data
Sample
  • Complex: Spliceosome immediately after branching
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsGalej WP / Wilkinson MF / Fica SM / Oubridge C / Newman AJ / Nagai K
CitationJournal: Nature / Year: 2016
Title: Cryo-EM structure of the spliceosome immediately after branching.
Authors: Wojciech P Galej / Max E Wilkinson / Sebastian M Fica / Chris Oubridge / Andrew J Newman / Kiyoshi Nagai /
Abstract: Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the ...Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5'-splice site is cleaved but remains close to the catalytic Mg site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5'-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2'OH. The 5'-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson-Crick interactions between the branch helix and 5'-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation.
History
DepositionJul 17, 2016-
Header (metadata) releaseJul 27, 2016-
Map releaseAug 3, 2016-
UpdateAug 30, 2017-
Current statusAug 30, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4059.png

Downloads & links

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Map

FileDownload / File: emd_4059.map.gz / Format: CCP4 / Size: 266.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.43 Å
Density
Contour LevelBy AUTHOR: 0.027 / Movie #1: 0.027
Minimum - Maximum-0.06751991 - 0.16137694
Average (Standard dev.)0.000012637265 (±0.0041432837)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions412412412
Spacing412412412
CellA=B=C: 589.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.431.431.43
M x/y/z412412412
origin x/y/z0.0000.0000.000
length x/y/z589.160589.160589.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS412412412
D min/max/mean-0.0680.1610.000

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Supplemental data

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Sample components

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Entire : Spliceosome immediately after branching

EntireName: Spliceosome immediately after branching
Components
  • Complex: Spliceosome immediately after branching

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Supramolecule #1: Spliceosome immediately after branching

SupramoleculeName: Spliceosome immediately after branching / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#35
Details: Splicing extract was prepared from Prp18-HA or Slu7-TAPS yeast strains. An in vitro transcribed yeast UBC4 pre-mRNA substrate (with 2 x MS2 bacteriophage coat protein-binding stem loops at ...Details: Splicing extract was prepared from Prp18-HA or Slu7-TAPS yeast strains. An in vitro transcribed yeast UBC4 pre-mRNA substrate (with 2 x MS2 bacteriophage coat protein-binding stem loops at the 5' end and with the 3'-splice site sequence UAGAG mutated to UACAC) was pre-bound to an MS2-maltose binding protein fusion protein. This substrate-protein complex was added to the splicing extract. The splicing reaction proceeded through the first step but the second step was blocked by the 3' splice site mutation. Substrate-bound spliceosomes from the splicing extract were purified on amylose resin and eluted with maltose. Subsequently the spliceosomes were captured on anti-HA-agarose (for Prp18-HA-tagged) or streptactin resin (for Slu7-TAPS tagged) and eluted with HA peptide or desthiobiotin, respectively. Purified spliceosomes were then dialysed against 20 mM HEPES KOH pH 7.8, 75 mM KCl, 0.25 mM EDTA
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

Concentration0.3 mg/mL
BufferpH: 7.8
Component:
ConcentrationNameFormula
20.0 millimolarHepes.KOH pH 7.8
75.0 millimolarpotassium chlorideKCl
250.0 micromolarEDTAEthylenediaminetetraacetic acid
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 6.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
Details: 3 microlitres sample were applied to the grid, left for 30 seconds and then blotted for 2.5-3.0 seconds before plunging..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 35714 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Quantum
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-20 / Number real images: 2213 / Average exposure time: 0.8 sec. / Average electron dose: 2.0 e/Å2
Details: Total dose: 40 electrons/Angstrom^2 over 16 seconds. 20 movie frames collected at 1.25 frames per second.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 248000
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: EMDB MAP
EMDB ID:

6413


Details: Intron-lariat spliceosome complex low-pass filtered to 60 Angstrom
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final 3D classificationSoftware - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 47484
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsUsed secondary structure restraints generated in ProSMART and LibG.
RefinementSpace: RECIPROCAL / Protocol: OTHER

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