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- EMDB-1294: Three-dimensional structure of the native spliceosome by cryo-ele... -

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Entry
Database: EMDB / ID: 1294
TitleThree-dimensional structure of the native spliceosome by cryo-electron microscopy.
Map dataBiological isosurface is at density value 0.007
Samplenative spliceosome:
SourceHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 22 Å resolution
AuthorsAzubel M / Wolf SG / Sperling J / Sperling R
CitationJournal: Mol. Cell / Year: 2004
Title: Three-dimensional structure of the native spliceosome by cryo-electron microscopy.
Authors: Maia Azubel / Sharon G Wolf / Joseph Sperling / Ruth Sperling
Abstract: Splicing of pre-mRNA occurs in a multicomponent macromolecular machine--the spliceosome. The spliceosome can be assembled in vitro by a stepwise assembly of a number of snRNPs and additional proteins ...Splicing of pre-mRNA occurs in a multicomponent macromolecular machine--the spliceosome. The spliceosome can be assembled in vitro by a stepwise assembly of a number of snRNPs and additional proteins on exogenously added pre-mRNA. In contrast, splicing in vivo occurs in preformed particles where endogenous pre-mRNAs are packaged with all five spliceosomal U snRNPs (penta-snRNP) together with other splicing factors. Here we present a three-dimensional image reconstruction by cryo-electron microscopy of native spliceosomes, derived from cell nuclei, at a resolution of 20 angstroms. The structure revealed an elongated globular particle made up of two distinct subunits connected to each other leaving a tunnel in between. We show here that the larger subunit is a suitable candidate to accommodate the penta-snRNP, and that the tunnel could accommodate the pre-mRNA component of the spliceosome. The features this structure reveals provide new insight into the global architecture of the native splicing machine.
DateDeposition: Sep 7, 2006 / Header (metadata) release: Nov 12, 2006 / Map release: Nov 12, 2006 / Last update: Oct 24, 2012

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00779575
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.00779575
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_1294.map.gz (map file in CCP4 format, 3908 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
100 pix
5.25 Å/pix.
= 525. Å
100 pix
5.25 Å/pix.
= 525. Å
100 pix
5.25 Å/pix.
= 525. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.25 Å
Density
Contour Level:0.00621, 0.0077958 (movie #1):
Minimum - Maximum-0.00948555 - 0.0274964
Average (Standard dev.)0.000361584 (0.00275396)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions100100100
Origin000
Limit999999
Spacing100100100
CellA=B=C: 525 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.255.255.25
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z525.000525.000525.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-0.0090.0270.000

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Supplemental data

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Sample components

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Entire native spliceosome

EntireName: native spliceosome / Number of components: 1 / Oligomeric State: monomeric
MassExperimental: 4.8 MDa / Measured by: STEM mass measurement

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Component #1: cellular-component, native spliceosome

Cellular-componentName: native spliceosome / Recombinant expression: No
MassTheoretical: 4.8 MDa
SourceSpecies: Homo sapiens (human)
Source (natural)Organelle: Nucleus / Location in cell: nucleus / Cell: HeLa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Support filmlacey (SPI) or Quantifoil grids
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 95 %
Method: the samples were incubated in Teflon wells under charged lipid monolayers for 20 min., picked up on grids, rinsed, blotted and plunged into liquid ethane.
Details: Vitrification instrument: I. Talmon plunger

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20
Details: magnification with calibrated camera postmag factor was 93,620
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 10 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 62000 X (nominal) / Astigmatism: correction at 100,000 to 250,000 times mag / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 4000 nm
Specimen HolderHolder: side entry liquid nitrogen-cooled cryo specimen holder
Model: GATAN LIQUID NITROGEN / Temperature: 97 K ( 95 - 100 K)
CameraDetector: GENERIC TVIPS

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Image acquisition

Image acquisitionSampling size: 24 microns / Bit depth: 16 / Details: TVIPS Biocam 1k X 1k CCD camera

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 7500 / Applied symmetry: C1 (asymmetric)
3D reconstructionAlgorithm: common lines / Software: Spider / CTF correction: phase correction, each particle / Resolution: 22 Å / Resolution method: FSC 0.5
Euler angles: see Supplemental material at Molecular Cell website.
Details: see Supplemental material at Molecular Cell website

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