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- EMDB-3688: Structure of a pre-catalytic spliceosome (B7 map) -

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Basic information

Entry
Database: EMDB / ID: EMD-3688
TitleStructure of a pre-catalytic spliceosome (B7 map)
Map data
SamplePre-catalytic B complex Spliceosome
Biological speciesSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsPlaschka C / Lin P-C / Nagai K
CitationJournal: Nature / Year: 2017
Title: Structure of a pre-catalytic spliceosome.
Authors: Clemens Plaschka / Pei-Chun Lin / Kiyoshi Nagai /
Abstract: Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome's catalytic centre. Here we report the cryo-electron microscopy ...Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome's catalytic centre. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 small nuclear ribonucleoprotein particle (snRNP) associates with U4/U6.U5 tri-snRNP through the U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2. The 3' region of the U2 snRNP is flexibly attached to the SF3b-containing domain and protrudes over the concave surface of tri-snRNP, where the U1 snRNP may reside before its release from the pre-mRNA 5' splice site. The U6 ACAGAGA sequence forms a hairpin that weakly tethers the 5' splice site. The B complex proteins Prp38, Snu23 and Spp381 bind the Prp8 N-terminal domain and stabilize U6 ACAGAGA stem-pre-mRNA and Brr2-U4 small nuclear RNA interactions. These results provide important insights into the events leading to active site formation.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionApr 24, 2017-
Header (metadata) releaseMay 31, 2017-
Map releaseMay 31, 2017-
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: 0.0348
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0348
  • 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_3688.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.43 Å/pix.
x 480 pix.
= 686.4 Å
1.43 Å/pix.
x 480 pix.
= 686.4 Å
1.43 Å/pix.
x 480 pix.
= 686.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.43 Å
Density
Contour LevelBy AUTHOR: 0.0348 / Movie #1: 0.0348
Minimum - Maximum-0.058982532 - 0.16124116
Average (Standard dev.)-0.000093418035 (±0.0046612304)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 686.39996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.431.431.43
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z686.400686.400686.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.0590.161-0.000

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Supplemental data

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Additional map: unsharpened B7 map

Fileemd_3688_additional.map
Annotationunsharpened B7 map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Pre-catalytic B complex Spliceosome

EntireName: Pre-catalytic B complex Spliceosome / Number of components: 1

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Component #1: protein, Pre-catalytic B complex Spliceosome

ProteinName: Pre-catalytic B complex Spliceosome / Recombinant expression: No
MassExperimental: 2.5 MDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.5 mg/mL / Buffer solution: Buffer pH: HEPES, 7.9; EDTA, 8.0 / pH: 7.9
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 100 %
Details: Grids were glow-discharged for 15 s before deposition of 3 microliter sample (~1.5 mg mL-1), and subsequently incubated for 2-3.5 s before blotting and vitrification by plunging into liquid ...Details: Grids were glow-discharged for 15 s before deposition of 3 microliter sample (~1.5 mg mL-1), and subsequently incubated for 2-3.5 s before blotting and vitrification by plunging into liquid ethane with a Vitrobot Mark III (FEI) operated at 4 degrees Celsius and 100% humidity..

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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: 56 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 81000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF Quantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 5115 / Sampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 62406
Details: Movies were binned once and aligned using MOTIONCORR.
3D reconstructionSoftware: RELION / Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF
Details: A temperature factor of -80 A2 was applied (sharpened B7 map).

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Atomic model buiding

Modeling #1Refinement space: REAL / Overall bvalue: 80

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