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- EMDB-13813: A-like U2 snRNP medium-resolution map -

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Open data


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Basic information

Entry
Database: EMDB / ID: EMD-13813
TitleA-like U2 snRNP medium-resolution map
Map data
Sample
  • Complex: 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: 2.6 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: 0.0199
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0199
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13813.png

Downloads & links

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Map

FileDownload / File: emd_13813.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.28 Å/pix.
x 320 pix.
= 409.6 Å		1.28 Å/pix.
x 320 pix.
= 409.6 Å		1.28 Å/pix.
x 320 pix.
= 409.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.28 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0199 / Movie #1: 0.0199
Minimum - Maximum-0.017098706 - 0.08184837
Average (Standard dev.)0.00012422318 (±0.0020224713)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 409.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.281.281.28
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z409.600409.600409.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0170.0820.000

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Supplemental data

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Mask #1

Fileemd_13813_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13813_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_13813_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 : A-like U2 snRNP

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

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Supramolecule #1: A-like U2 snRNP

SupramoleculeName: 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 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 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 KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 15681 / Average exposure time: 1.0 sec. / Average electron dose: 52.03 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1470005
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15) / Software - details: Ab-initio reconstruction
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 658325
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7q3l

chain_id: A, source_name: PDB, initial_model_type: experimental model

7q3l

chain_id: B, source_name: PDB, initial_model_type: experimental model

7q3l

chain_id: C, source_name: PDB, initial_model_type: experimental model

7q3l

chain_id: E, source_name: PDB, initial_model_type: experimental model

3lqv

chain_id: F, residue_range: 12-101, source_name: PDB, initial_model_type: experimental model

7q3l

chain_id: G, source_name: PDB, initial_model_type: experimental model

7abh

chain_id: 1, residue_range: 42-85, source_name: PDB, initial_model_type: experimental model

7q3l

chain_id: 2, source_name: PDB, initial_model_type: experimental model

7q3l

chain_id: 9, source_name: PDB, initial_model_type: experimental model
RefinementSpace: RECIPROCAL

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