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- EMDB-13814: AMP-PCP-treated A-like U2 snRNP -

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Basic information

Entry
Database: EMDB / ID: EMD-13814
TitleAMP-PCP-treated A-like U2 snRNP
Map data
Sample
  • Complex: AMP-PCP-treated A-like U2 snRNP
    • Complex: A-like U2 snRNP
    • Complex: BPS Oligo
KeywordsSplicing / snRNP / Spliceosome / NUCLEAR PROTEIN
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsTholen J / Galej WP
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)950278European Union
CitationJournal: Science / Year: 2022
Title: Structural basis of branch site recognition by the human spliceosome.
Authors: Jonas Tholen / Michal Razew / Felix Weis / Wojciech P Galej /
Abstract: Recognition of the intron branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is a critical event during spliceosome assembly. In mammals, BS sequences are poorly conserved, and ...Recognition of the intron branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is a critical event during spliceosome assembly. In mammals, BS sequences are poorly conserved, and unambiguous intron recognition cannot be achieved solely through a base-pairing mechanism. We isolated human 17 U2 snRNP and reconstituted in vitro its adenosine 5´-triphosphate (ATP)–dependent remodeling and binding to the pre–messenger RNA substrate. We determined a series of high-resolution (2.0 to 2.2 angstrom) structures providing snapshots of the BS selection process. The substrate-bound U2 snRNP shows that SF3B6 stabilizes the BS:U2 snRNA duplex, which could aid binding of introns with poor sequence complementarity. ATP-dependent remodeling uncoupled from substrate binding captures U2 snRNA in a conformation that competes with BS recognition, providing a selection mechanism based on branch helix stability.
History
DepositionNov 1, 2021-
Header (metadata) releaseMar 2, 2022-
Map releaseMar 2, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.238
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.238
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13814.png

Downloads & links

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Map

FileDownload / File: emd_13814.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.94 Å/pix.
x 450 pix.
= 423. Å		0.94 Å/pix.
x 450 pix.
= 423. Å		0.94 Å/pix.
x 450 pix.
= 423. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.94 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.238 / Movie #1: 0.238
Minimum - Maximum-0.6492927 - 1.6346929
Average (Standard dev.)-0.00030375912 (±0.02888674)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 423.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.940.940.94
M x/y/z450450450
origin x/y/z0.0000.0000.000
length x/y/z423.000423.000423.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS450450450
D min/max/mean-0.6491.635-0.000

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Supplemental data

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Half map: #1

Fileemd_13814_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_13814_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 : AMP-PCP-treated A-like U2 snRNP

EntireName: AMP-PCP-treated A-like U2 snRNP
Components
  • Complex: AMP-PCP-treated A-like U2 snRNP
    • Complex: A-like U2 snRNP
    • Complex: BPS Oligo

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Supramolecule #1: AMP-PCP-treated A-like U2 snRNP

SupramoleculeName: AMP-PCP-treated A-like U2 snRNP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
Details: Spliceosomal complex A-like U2 snRNP generated by binding BPS oligo in the presence of AMP-PCP to 17S U2 snRNP that was isolated from nuclear extract of HEK293F cells by affinity chromatography.
Molecular weightTheoretical: 300 KDa

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Supramolecule #2: A-like U2 snRNP

SupramoleculeName: A-like U2 snRNP / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#9
Details: Spliceosomal complex A-like U2 snRNP generated by binding BPS oligo in the presence of AMP-PCP to 17S U2 snRNP that was isolated from nuclear extract of HEK293F cells by affinity chromatography.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: BPS Oligo

SupramoleculeName: BPS Oligo / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #10 / Details: BPS oligo
Source (natural)Organism: synthetic construct (others)

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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

Concentration0.3 mg/mL
BufferpH: 7.9
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMKClPotassium chloride
2.0 mMMgCl2Magnesium chloride

Details: Sample after desalting may have also contained up to 5% glycerol.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 2230 / Average electron dose: 47.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 150000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 320883
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 63915
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15) / Software - details: Ab-initio reconstruction
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 2.15)

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