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- EMDB-4672: Human U5.U4/U6 tri-snRNP, multi-body refinement, Brr2 map -

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Basic information

Entry
Database: EMDB / ID: EMD-4672
TitleHuman U5.U4/U6 tri-snRNP, multi-body refinement, Brr2 map
Map dataHuman U5.U4/U6 tri-snRNP, multi-body refinement, Brr2 map
Sample
  • Complex: Human U4/U6.U5 tri-snRNP
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsCharenton C / Wilkinson ME / Nagai K
CitationJournal: Science / Year: 2019
Title: Mechanism of 5' splice site transfer for human spliceosome activation.
Authors: Clément Charenton / Max E Wilkinson / Kiyoshi Nagai /
Abstract: The prespliceosome, comprising U1 and U2 small nuclear ribonucleoproteins (snRNPs) bound to the precursor messenger RNA 5' splice site (5'SS) and branch point sequence, associates with the U4/U6.U5 ...The prespliceosome, comprising U1 and U2 small nuclear ribonucleoproteins (snRNPs) bound to the precursor messenger RNA 5' splice site (5'SS) and branch point sequence, associates with the U4/U6.U5 tri-snRNP to form the fully assembled precatalytic pre-B spliceosome. Here, we report cryo-electron microscopy structures of the human pre-B complex captured before U1 snRNP dissociation at 3.3-angstrom core resolution and the human tri-snRNP at 2.9-angstrom resolution. U1 snRNP inserts the 5'SS-U1 snRNA helix between the two RecA domains of the Prp28 DEAD-box helicase. Adenosine 5'-triphosphate-dependent closure of the Prp28 RecA domains releases the 5'SS to pair with the nearby U6 ACAGAGA-box sequence presented as a mobile loop. The structures suggest that formation of the 5'SS-ACAGAGA helix triggers remodeling of an intricate protein-RNA network to induce Brr2 helicase relocation to its loading sequence in U4 snRNA, enabling Brr2 to unwind the U4/U6 snRNA duplex to allow U6 snRNA to form the catalytic center of the spliceosome.
History
DepositionMar 8, 2019-
Header (metadata) releaseApr 24, 2019-
Map releaseApr 24, 2019-
UpdateMay 8, 2019-
Current statusMay 8, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4672.png

Downloads & links

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Map

FileDownload / File: emd_4672.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman U5.U4/U6 tri-snRNP, multi-body refinement, Brr2 map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.02 Å/pix.
x 420 pix.
= 429.24 Å		1.02 Å/pix.
x 420 pix.
= 429.24 Å		1.02 Å/pix.
x 420 pix.
= 429.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.022 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.08317234 - 0.1480678
Average (Standard dev.)0.0000020449563 (±0.0025835454)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 429.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0221.0221.022
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z429.240429.240429.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS420420420
D min/max/mean-0.0830.1480.000

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Supplemental data

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Sample components

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Entire : Human U4/U6.U5 tri-snRNP

EntireName: Human U4/U6.U5 tri-snRNP
Components
  • Complex: Human U4/U6.U5 tri-snRNP

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Supramolecule #1: Human U4/U6.U5 tri-snRNP

SupramoleculeName: Human U4/U6.U5 tri-snRNP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#32
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.7 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

Concentration1.3 mg/mL
BufferpH: 7.9
Component:
ConcentrationName
20.0 mMHepes KOH
50.0 mMKCl
1.0 mMDTT
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III / Details: Wait 30s, blot for 2s to 3s..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Average exposure time: 6.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 130000
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: 1147653
CTF correctionSoftware - Name: Gctf
Startup modelType of model: EMDB MAP
EMDB ID:

6581

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 585488
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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