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- EMDB-4236: Human Bact spliceosome state 4 unmasked -

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Basic information

Entry
Database: EMDB / ID: EMD-4236
TitleHuman Bact spliceosome state 4 unmasked
Map data
Sample
  • Complex: human Bact spliceosome state 1 unmasked
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsHaselbach D / Komarov I / Agafonov D / Kastner B / Luehrmann R / Stark H
CitationJournal: Cell / Year: 2018
Title: Structure and Conformational Dynamics of the Human Spliceosomal B Complex.
Authors: David Haselbach / Ilya Komarov / Dmitry E Agafonov / Klaus Hartmuth / Benjamin Graf / Olexandr Dybkov / Henning Urlaub / Berthold Kastner / Reinhard Lührmann / Holger Stark /
Abstract: The spliceosome is a highly dynamic macromolecular complex that precisely excises introns from pre-mRNA. Here we report the cryo-EM 3D structure of the human B spliceosome at 3.4 Å resolution. In ...The spliceosome is a highly dynamic macromolecular complex that precisely excises introns from pre-mRNA. Here we report the cryo-EM 3D structure of the human B spliceosome at 3.4 Å resolution. In the B state, the spliceosome is activated but not catalytically primed, so that it is functionally blocked prior to the first catalytic step of splicing. The spliceosomal core is similar to the yeast B spliceosome; important differences include the presence of the RNA helicase aquarius and peptidyl prolyl isomerases. To examine the overall dynamic behavior of the purified spliceosome, we developed a principal component analysis-based approach. Calculating the energy landscape revealed eight major conformational states, which we refined to higher resolution. Conformational differences of the highly flexible structural components between these eight states reveal how spliceosomal components contribute to the assembly of the spliceosome, allowing it to generate a dynamic interaction network required for its subsequent catalytic activation.
History
DepositionDec 22, 2017-
Header (metadata) releaseFeb 7, 2018-
Map releaseFeb 7, 2018-
UpdateFeb 7, 2018-
Current statusFeb 7, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0148
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0148
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4236.png

Downloads & links

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Map

FileDownload / File: emd_4236.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.16 Å/pix.
x 420 pix.
= 487.2 Å		1.16 Å/pix.
x 420 pix.
= 487.2 Å		1.16 Å/pix.
x 420 pix.
= 487.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.16 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0148 / Movie #1: 0.0148
Minimum - Maximum-0.016119761 - 0.061895665
Average (Standard dev.)0.0007494811 (±0.0041112616)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 487.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.161.161.16
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z487.200487.200487.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-128-128-128
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS420420420
D min/max/mean-0.0160.0620.001

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Supplemental data

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Sample components

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Entire : human Bact spliceosome state 1 unmasked

EntireName: human Bact spliceosome state 1 unmasked
Components
  • Complex: human Bact spliceosome state 1 unmasked

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Supramolecule #1: human Bact spliceosome state 1 unmasked

SupramoleculeName: human Bact spliceosome state 1 unmasked / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Strain: HELA
Molecular weightTheoretical: 4.5 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

Concentration0.05 mg/mL
BufferpH: 7.9
Component:
ConcentrationNameFormula
20.0 mMHEPES
150.0 mMNaCl
1.5 mMMgCl2
GridModel: Quantifoil R3.5/1 / Material: COPPER / Mesh: 400 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: HOLEY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 277 K / Instrument: LEICA EM GP / Details: blot with blotting sensor.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Alignment procedureComa free - Residual tilt: 14.0 mrad
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs corrector with two hexapoles elements
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Number real images: 32000 / Average exposure time: 1.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 0.001 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3300000
CTF correctionSoftware - Name: Gctf
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 156794
Initial angle assignmentType: OTHER / Software - Name: EMAN2
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final 3D classificationNumber classes: 220 / Software - Name: RELION

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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