|Entry||Database: EMDB / ID: 1294|
|Title||Three-dimensional structure of the native spliceosome by cryo-electron microscopy.|
|Map data||Biological isosurface is at density value 0.007|
|Source||Homo sapiens (human)|
|Method||single particle reconstruction / cryo EM / 22 Å resolution|
|Authors||Azubel M / Wolf SG / Sperling J / Sperling R|
|Citation||Journal: 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.
|Date||Deposition: Sep 7, 2006 / Header (metadata) release: Nov 12, 2006 / Map release: Nov 12, 2006 / Last update: Oct 24, 2012|
|Structure viewer||EM map: |
Downloads & links
|File||emd_1294.map.gz (map file in CCP4 format, 3908 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 5.25 Å|
CCP4 map header:
-Entire native spliceosome
|Entire||Name: native spliceosome / Number of components: 1 / Oligomeric State: monomeric|
|Mass||Experimental: 4.8 MDa / Measured by: STEM mass measurement|
-Component #1: cellular-component, native spliceosome
|Cellular-component||Name: native spliceosome / Recombinant expression: No|
|Mass||Theoretical: 4.8 MDa|
|Source||Species: Homo sapiens (human)|
|Source (natural)||Organelle: Nucleus / Location in cell: nucleus / Cell: HeLa|
|Specimen||Specimen state: particle / Method: cryo EM|
|Support film||lacey (SPI) or Quantifoil grids|
|Vitrification||Instrument: 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
-Electron microscopy imaging
Model: Tecnai F20 / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI F20|
Details: magnification with calibrated camera postmag factor was 93,620
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 10 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 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 Holder||Holder: side entry liquid nitrogen-cooled cryo specimen holder|
Model: GATAN LIQUID NITROGEN / Temperature: 97 K ( 95 - 100 K)
|Camera||Detector: GENERIC TVIPS|
|Image acquisition||Sampling size: 24 microns / Bit depth: 16 / Details: TVIPS Biocam 1k X 1k CCD camera|
|Processing||Method: single particle reconstruction / Number of projections: 7500 / Applied symmetry: C1 (asymmetric)|
|3D reconstruction||Algorithm: 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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