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- EMDB-3682: Structure of a pre-catalytic spliceosome (B1 map) -

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Basic information

Entry
Database: EMDB / ID: EMD-3682
TitleStructure of a pre-catalytic spliceosome (B1 map)
Map datasharpened B1 map
Sample
  • Complex: Pre-catalytic B complex Spliceosome
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 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.
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: 0.038
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.038
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3682.png

Downloads & links

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Map

FileDownload / File: emd_3682.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened B1 map
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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.038 / Movie #1: 0.038
Minimum - Maximum-0.07190707 - 0.16152641
Average (Standard dev.)-0.00022361842 (±0.0028545337)
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-0.0720.162-0.000

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Supplemental data

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Additional map: unsharpened B1 map

Fileemd_3682_additional.map
Annotationunsharpened B1 map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Pre-catalytic B complex Spliceosome

EntireName: Pre-catalytic B complex Spliceosome
Components
  • Complex: Pre-catalytic B complex Spliceosome

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Supramolecule #1: Pre-catalytic B complex Spliceosome

SupramoleculeName: Pre-catalytic B complex Spliceosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BCY123
Molecular weightExperimental: 2.5 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

Concentration1.5 mg/mL
BufferpH: 7.9
Component:
ConcentrationFormula
20.0 mMHEPES
50.0 mMKCl
0.2 mMEDTA
1.0 mMDTT
2.5 mMDesthiobiotin

Details: Buffer pH: HEPES, 7.9; EDTA, 8.0
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK III
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

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-20 / Number real images: 5115 / Average exposure time: 16.0 sec. / Average electron dose: 56.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 5.3 µm / Calibrated defocus min: 0.35 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsMovies were binned once and aligned using MOTIONCORR.
Particle selectionNumber selected: 496581
CTF correctionSoftware:
Namedetails
CTFFIND (ver. 4.1)CTF determination
RELION (ver. 2.0)CTF correction
Startup modelType of model: OTHER
Details: The negative stain map of human B-delta-U1 (EMD-1066) was used as an initial reference for three-dimensional refinement of a 12229 particle negative stain data set of the yeast B complex. A ...Details: The negative stain map of human B-delta-U1 (EMD-1066) was used as an initial reference for three-dimensional refinement of a 12229 particle negative stain data set of the yeast B complex. A single round of 3D classification revealed a B complex density from 3707 particles that was refined to an estimated resolution of 50 A. This density was low-pass filtered to 60 A and used for processing of cryo-EM data.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0)
Details: A temperature factor of -125 A2 was applied (sharpened B1 map).
Number images used: 254095
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final 3D classificationSoftware - Name: RELION (ver. 2.0)

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Atomic model buiding 1

RefinementSpace: REAL / Overall B value: 125

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