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

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Basic information

Entry
Database: EMDB / ID: EMD-1294
TitleThree-dimensional structure of the native spliceosome by cryo-electron microscopy.
Map dataBiological isosurface is at density value 0.007
Sample
  • Sample: native spliceosome
  • Organelle or cellular component: native spliceosome
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 22.0 Å
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.
History
DepositionSep 7, 2006-
Header (metadata) releaseNov 12, 2006-
Map releaseNov 12, 2006-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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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
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1294.gif

Downloads & links

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Map

FileDownload / File: emd_1294.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBiological isosurface is at density value 0.007
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour Level1: 0.00621 / Movie #1: 0.0077958
Minimum - Maximum-0.00948555 - 0.0274964
Average (Standard dev.)0.000361584 (±0.00275396)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 525 Å
α=β=γ: 90 °

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
Components
  • Sample: native spliceosome
  • Organelle or cellular component: native spliceosome

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Supramolecule #1000: native spliceosome

SupramoleculeName: native spliceosome / type: sample / ID: 1000 / Oligomeric state: monomeric / Number unique components: 1
Molecular weightExperimental: 4.8 MDa / Method: STEM mass measurement

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Supramolecule #1: native spliceosome

SupramoleculeName: native spliceosome / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus / Location in cell: nucleus
Molecular weightTheoretical: 4.8 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

GridDetails: lacey (SPI) or Quantifoil grids
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: I. Talmon plunger
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.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 62000
Sample stageSpecimen holder: side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 95 K / Max: 100 K / Average: 97 K
Alignment procedureLegacy - Astigmatism: correction at 100,000 to 250,000 times mag
Detailsmagnification with calibrated camera postmag factor was 93,620
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS / Digitization - Sampling interval: 24 µm / Average electron dose: 10 e/Å2 / Details: TVIPS Biocam 1k X 1k CCD camera / Bits/pixel: 16
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: phase correction, each particle
Final angle assignmentDetails: see Supplemental material at Molecular Cell website.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider
Details: see Supplemental material at Molecular Cell website
Number images used: 7500

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