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- PDB-7q4p: U2 snRNP after ATP-dependent remodelling -

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Basic information

Entry
Database: PDB / ID: 7q4p
TitleU2 snRNP after ATP-dependent remodelling
Components
  • (Splicing factor 3A subunit ...) x 2
  • (Splicing factor 3B subunit ...) x 4
  • PHD finger-like domain-containing protein 5A
  • U2 snRNA
KeywordsNUCLEAR PROTEIN / snRNP / Spliceosome / U2 snRNP / Splicing
Function / homology
Function and homology information


U11/U12 snRNP / B-WICH complex / splicing factor binding / U12-type spliceosomal complex / RNA splicing, via transesterification reactions / U2-type spliceosomal complex / U2-type precatalytic spliceosome / U2-type prespliceosome assembly / SAGA complex / U2 snRNP ...U11/U12 snRNP / B-WICH complex / splicing factor binding / U12-type spliceosomal complex / RNA splicing, via transesterification reactions / U2-type spliceosomal complex / U2-type precatalytic spliceosome / U2-type prespliceosome assembly / SAGA complex / U2 snRNP / positive regulation of transcription by RNA polymerase III / U2-type prespliceosome / positive regulation of transcription by RNA polymerase I / precatalytic spliceosome / spliceosomal complex assembly / mRNA Splicing - Minor Pathway / regulation of RNA splicing / mRNA 3'-splice site recognition / U2 snRNA binding / regulation of DNA repair / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / RNA splicing / stem cell differentiation / spliceosomal complex / B-WICH complex positively regulates rRNA expression / negative regulation of protein catabolic process / mRNA processing / nuclear matrix / positive regulation of neuron projection development / mRNA splicing, via spliceosome / nuclear speck / chromatin remodeling / mRNA binding / protein-containing complex binding / nucleolus / positive regulation of DNA-templated transcription / positive regulation of transcription by RNA polymerase II / DNA binding / RNA binding / zinc ion binding / nucleoplasm / nucleus
Similarity search - Function
Splicing factor SF3a60 binding domain / Splicing factor SF3a60 binding domain / : / Replication stress response SDE2 C-terminal / Cactus-binding C-terminus of cactin protein / SF3A2 domain / Pre-mRNA-splicing factor SF3a complex subunit 2 (Prp11) / Splicing factor SF3a60 /Prp9 subunit, C-terminal / SF3A3 domain / SF3a60/Prp9 C-terminal ...Splicing factor SF3a60 binding domain / Splicing factor SF3a60 binding domain / : / Replication stress response SDE2 C-terminal / Cactus-binding C-terminus of cactin protein / SF3A2 domain / Pre-mRNA-splicing factor SF3a complex subunit 2 (Prp11) / Splicing factor SF3a60 /Prp9 subunit, C-terminal / SF3A3 domain / SF3a60/Prp9 C-terminal / Pre-mRNA-splicing factor SF3A3, of SF3a complex, Prp9 / Splicing factor 3B, subunit 5 / Splicing factor 3B subunit 1 / Splicing factor 3B subunit 1 / Domain of unknown function DUF382 / Domain of unknown function (DUF382) / Zinc-finger of C2H2 type / PHF5-like / PHF5-like protein / PSP, proline-rich / PSP / proline-rich domain in spliceosome associated proteins / Splicing factor 3B subunit 5/RDS3 complex subunit 10 / Splicing factor 3B subunit 10 (SF3b10) / Splicing factor 3B subunit 1-like / Matrin/U1-C, C2H2-type zinc finger / Zinc finger matrin-type profile. / SAP domain / SAP motif profile. / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / Matrin/U1-C-like, C2H2-type zinc finger / U1-like zinc finger / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Mono-functional DNA-alkylating methyl methanesulfonate N-term / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / CPSF A subunit region / Zinc finger C2H2 superfamily / Zinc finger C2H2-type / Armadillo-like helical / Armadillo-type fold / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA (> 100) / Splicing factor 3B subunit 1 / Splicing factor 3A subunit 3 / Splicing factor 3B subunit 2 / Splicing factor 3B subunit 3 / Splicing factor 3A subunit 2 / PHD finger-like domain-containing protein 5A / Splicing factor 3B subunit 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.15 Å
AuthorsTholen, J. / Galej, W.P.
Funding supportEuropean Union, 1items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 30, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 6, 2022Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.2Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model / refine
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: Splicing factor 3A subunit 2
2: U2 snRNA
9: Splicing factor 3A subunit 3
A: Splicing factor 3B subunit 1
B: Splicing factor 3B subunit 2
C: Splicing factor 3B subunit 3
E: Splicing factor 3B subunit 5
G: PHD finger-like domain-containing protein 5A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)573,55813
Polymers573,2318
Non-polymers3275
Water4,342241
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area35900 Å2
ΔGint-101 kcal/mol
Surface area62170 Å2

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Components

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Splicing factor 3A subunit ... , 2 types, 2 molecules 19

#1: Protein Splicing factor 3A subunit 2 / SF3a66 / Spliceosome-associated protein 62 / SAP 62


Mass: 49327.355 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293 / References: UniProt: Q15428
#3: Protein Splicing factor 3A subunit 3 / SF3a60 / Spliceosome-associated protein 61 / SAP 61


Mass: 58934.844 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: Q12874

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Splicing factor 3B subunit ... , 4 types, 4 molecules ABCE

#4: Protein Splicing factor 3B subunit 1 / Pre-mRNA-splicing factor SF3b 155 kDa subunit / SF3b155 / Spliceosome-associated protein 155 / SAP 155


Mass: 146024.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: O75533
#5: Protein Splicing factor 3B subunit 2 / Pre-mRNA-splicing factor SF3b 145 kDa subunit / SF3b145 / Spliceosome-associated protein 145 / SAP 145


Mass: 100377.812 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: Q13435
#6: Protein Splicing factor 3B subunit 3 / Pre-mRNA-splicing factor SF3b 130 kDa subunit / SF3b130 / STAF130 / Spliceosome-associated protein ...Pre-mRNA-splicing factor SF3b 130 kDa subunit / SF3b130 / STAF130 / Spliceosome-associated protein 130 / SAP 130


Mass: 135718.844 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: Q15393
#7: Protein Splicing factor 3B subunit 5 / SF3b5 / Pre-mRNA-splicing factor SF3b 10 kDa subunit


Mass: 10149.369 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: Q9BWJ5

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RNA chain / Protein , 2 types, 2 molecules 2G

#2: RNA chain U2 snRNA


Mass: 60270.602 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: GenBank: NR_002716
#8: Protein PHD finger-like domain-containing protein 5A / PHD finger-like domain protein 5A / Splicing factor 3B-associated 14 kDa protein / SF3b14b


Mass: 12427.524 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: Q7RTV0

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Non-polymers , 2 types, 246 molecules

#9: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 241 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Remodelled U2 snRNP / Type: COMPLEX
Details: Remodelled U2 snRNP obtained after incubation of 17S U2 snRNP with ATP. 17S U2 snRNP was isolated from nuclear extract of HEK293F cells by affinity chromatography.
Entity ID: #1-#8 / Source: NATURAL
Molecular weightValue: 1.08 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.9
Details: Sample after desalting may have also contained up to 5% glycerol.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMPotassium chlorideKCl1
32 mMMagnesium chlorideMgCl21
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 53.45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 15531
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement
EM software
IDNameVersionCategoryDetails
1Warp0.9particle selection
2SerialEMimage acquisition
4CTFFIND4.1.14CTF correction
7Coot0.9.4.1model fitting
8UCSF ChimeraX1.1model fitting
9ISOLDE1.1model fitting
11cryoSPARC2.15initial Euler assignmentAb-initio reconstruction
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
15REFMAC5model refinement
16LIBGmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1500165
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 158286 / Symmetry type: POINT
Atomic model buildingSpace: RECIPROCAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDPdb chain residue range
17Q3L

7q3l

A7Q3L1
27Q3L

7q3l

B7Q3L1
37Q3L

7q3l

C7Q3L1
47Q3L

7q3l

E7Q3L1
57Q3L

7q3l

G7Q3L1
67Q3L

7q3l

97Q3L1
77ABH

7abh

17ABH242-85
87Q3L

7q3l

27Q3L1
RefinementResolution: 2.15→2.15 Å / Cor.coef. Fo:Fc: 0.897 / SU B: 5.93 / SU ML: 0.14 / ESU R: 0.177
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.32815 --
obs0.32815 277730 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 60.241 Å2
Baniso -1Baniso -2Baniso -3
1-0.09 Å20.89 Å20.23 Å2
2--0.81 Å2-0.11 Å2
3----0.9 Å2
Refinement stepCycle: 1 / Total: 13456
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.01213610
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.7741.68318632
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.5045.3171624
ELECTRON MICROSCOPYr_dihedral_angle_2_deg37.93522.343683
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.484152131
ELECTRON MICROSCOPYr_dihedral_angle_4_deg15.1821580
ELECTRON MICROSCOPYr_chiral_restr0.1220.2011827
ELECTRON MICROSCOPYr_gen_planes_refined0.0120.0210075
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it0.8585.7236133
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it1.6198.5727647
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it0.5756.3447477
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined7.24758135
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.2→2.257 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.864 20595 -
obs--100 %

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