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- EMDB-3547: Cryo-EM structure of a human spliceosome activated for step 2 of ... -

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Basic information

Entry
Database: EMDB / ID: EMD-3547
TitleCryo-EM structure of a human spliceosome activated for step 2 of splicing (C* complex)
Map data
Sample
  • Complex: Human C* Spliceosome, unmasked refinement
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.4 Å
AuthorsBertram K / Liu WT
CitationJournal: Nature / Year: 2017
Title: Cryo-EM structure of a human spliceosome activated for step 2 of splicing.
Authors: Karl Bertram / Dmitry E Agafonov / Wen-Ti Liu / Olexandr Dybkov / Cindy L Will / Klaus Hartmuth / Henning Urlaub / Berthold Kastner / Holger Stark / Reinhard Lührmann /
Abstract: Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human ...Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2-U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 Å, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains. RNA helicase PRP22 is located about 100 Å from the catalytic centre, suggesting that it destabilizes the spliced mRNA after step two from a distance. Comparison of the structure of the yeast C and human C* complexes reveals numerous RNP rearrangements that are likely to be facilitated by PRP16, including a large-scale movement of the U2 small nuclear RNP.
History
DepositionDec 20, 2016-
Header (metadata) releaseJan 25, 2017-
Map releaseJan 25, 2017-
UpdateSep 20, 2017-
Current statusSep 20, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3547.png

Downloads & links

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Map

FileDownload / File: emd_3547.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.59 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.17935593 - 0.29795972
Average (Standard dev.)-0.00006143348 (±0.009490349)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 572.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.591.591.59
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z572.400572.400572.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.1790.298-0.000

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Supplemental data

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Half map: #1

Fileemd_3547_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_3547_half_map_2.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 : Human C* Spliceosome, unmasked refinement

EntireName: Human C* Spliceosome, unmasked refinement
Components
  • Complex: Human C* Spliceosome, unmasked refinement

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Supramolecule #1: Human C* Spliceosome, unmasked refinement

SupramoleculeName: Human C* Spliceosome, unmasked refinement / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#41
Source (natural)Organism: Homo sapiens (human)

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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

BufferpH: 6.4
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Digitization - Frames/image: 2-17 / Average electron dose: 2.1 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Reconstruction from negatively stained particles
Initial angle assignmentType: COMMON LINE
Final angle assignmentType: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 136534

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Atomic model buiding 1

RefinementSpace: REAL

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