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- EMDB-3768: Cryo-EM structure of a pre-catalytic human spliceosome primed for... -

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Entry
Database: EMDB / ID: EMD-3768
TitleCryo-EM structure of a pre-catalytic human spliceosome primed for activation (B complex). Unmasked refinement, sharpened map
Map datasharpened, unmasked refinement. Map was aligned to high resolution map (EMDB 3766) and RESAMPLED from 256 px boxsize to 432 px boxsize in Chimera to fit high-resolution map.
Sample
  • Complex: Cryo-EM structure of a pre-catalytic human spliceosome primed for activation (B complex)
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsBertram K
CitationJournal: Cell / Year: 2017
Title: Cryo-EM Structure of a Pre-catalytic Human Spliceosome Primed for Activation.
Authors: Karl Bertram / Dmitry E Agafonov / Olexandr Dybkov / David Haselbach / Majety N Leelaram / Cindy L Will / Henning Urlaub / Berthold Kastner / Reinhard Lührmann / Holger Stark /
Abstract: Little is known about the spliceosome's structure before its extensive remodeling into a catalytically active complex. Here, we report a 3D cryo-EM structure of a pre-catalytic human spliceosomal B ...Little is known about the spliceosome's structure before its extensive remodeling into a catalytically active complex. Here, we report a 3D cryo-EM structure of a pre-catalytic human spliceosomal B complex. The U2 snRNP-containing head domain is connected to the B complex main body via three main bridges. U4/U6.U5 tri-snRNP proteins, which are located in the main body, undergo significant rearrangements during tri-snRNP integration into the B complex. These include formation of a partially closed Prp8 conformation that creates, together with Dim1, a 5' splice site (ss) binding pocket, displacement of Sad1, and rearrangement of Brr2 such that it contacts its U4/U6 substrate and is poised for the subsequent spliceosome activation step. The molecular organization of several B-specific proteins suggests that they are involved in negatively regulating Brr2, positioning the U6/5'ss helix, and stabilizing the B complex structure. Our results indicate significant differences between the early activation phase of human and yeast spliceosomes.
History
DepositionJun 20, 2017-
Header (metadata) releaseAug 16, 2017-
Map releaseAug 16, 2017-
UpdateAug 23, 2017-
Current statusAug 23, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3768.png

Downloads & links

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Map

FileDownload / File: emd_3768.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened, unmasked refinement. Map was aligned to high resolution map (EMDB 3766) and RESAMPLED from 256 px boxsize to 432 px boxsize in Chimera to fit high-resolution map.
Voxel sizeX=Y=Z: 1.16 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.06067226 - 0.20512854
Average (Standard dev.)-0.00024091067 (±0.013150235)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 501.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.161.161.16
M x/y/z432432432
origin x/y/z0.0000.0000.000
length x/y/z501.120501.120501.120
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS432432432
D min/max/mean-0.0610.205-0.000

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Supplemental data

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Additional map: sharpened, unmasked refinement. Original, not resampled map

Fileemd_3768_additional.map
Annotationsharpened, unmasked refinement. Original, not resampled 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 : Cryo-EM structure of a pre-catalytic human spliceosome primed for...

EntireName: Cryo-EM structure of a pre-catalytic human spliceosome primed for activation (B complex)
Components
  • Complex: Cryo-EM structure of a pre-catalytic human spliceosome primed for activation (B complex)

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Supramolecule #1: Cryo-EM structure of a pre-catalytic human spliceosome primed for...

SupramoleculeName: Cryo-EM structure of a pre-catalytic human spliceosome primed for activation (B complex)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#43
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: 7.9
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
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.0 µm / Digitization - Frames/image: 2-17 / Average exposure time: 0.05 sec. / Average electron dose: 1.5 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 reconstructionResolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 44629
DetailsFALCON III Direct Electron Detector

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Atomic model buiding 1

RefinementSpace: REAL

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