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- PDB-7q4o: Substrate-bound A-like U2 snRNP -

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Basic information

Entry
Database: PDB / ID: 7q4o
TitleSubstrate-bound A-like U2 snRNP
Components
  • (Splicing factor 3A subunit ...) x 2
  • (Splicing factor 3B subunit ...) x 5
  • BPS oligo
  • PHD finger-like domain-containing protein 5A
  • U2 snRNAU2 spliceosomal RNA
KeywordsNUCLEAR PROTEIN / Splicing / snRNP / Spliceosome
Function / homology
Function and homology information


U11/U12 snRNP / B-WICH complex / splicing factor binding / U12-type spliceosomal complex / RNA splicing, via transesterification reactions / blastocyst formation / : / U2-type spliceosomal complex / U2-type precatalytic spliceosome / U2-type prespliceosome assembly ...U11/U12 snRNP / B-WICH complex / splicing factor binding / U12-type spliceosomal complex / RNA splicing, via transesterification reactions / blastocyst formation / : / U2-type spliceosomal complex / U2-type precatalytic spliceosome / U2-type prespliceosome assembly / U2 snRNP / SAGA complex / positive regulation of transcription by RNA polymerase III / U2-type prespliceosome / precatalytic spliceosome / spliceosomal complex assembly / positive regulation of transcription by RNA polymerase I / mRNA Splicing - Minor Pathway / regulation of RNA splicing / mRNA 3'-splice site recognition / regulation of DNA repair / U2 snRNA binding / 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 / positive regulation of neuron projection development / mRNA splicing, via spliceosome / mRNA processing / nuclear matrix / 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
SF3B6, RNA recognition motif / 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 ...SF3B6, RNA recognition motif / 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) / Matrin/U1-C, C2H2-type zinc finger / Splicing factor 3B subunit 1-like / 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 / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Armadillo-like helical / Armadillo-type fold / Nucleotide-binding alpha-beta plait domain superfamily / 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 ...: / 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 / Splicing factor 3B subunit 6
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.1 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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
F: Splicing factor 3B subunit 6
G: PHD finger-like domain-containing protein 5A
h: BPS oligo
hetero molecules


Theoretical massNumber of molelcules
Total (without water)593,51815
Polymers593,19110
Non-polymers3275
Water5,134285
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area39150 Å2
ΔGint-168 kcal/mol
Surface area105560 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: HEK293F / 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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RNA chain , 2 types, 2 molecules 2h

#2: RNA chain U2 snRNA / U2 spliceosomal RNA


Mass: 60228.523 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: GenBank: NR_002716
#10: RNA chain BPS oligo


Mass: 5395.286 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Splicing factor 3B subunit ... , 5 types, 5 molecules ABCEF

#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
#8: Protein Splicing factor 3B subunit 6 / Pre-mRNA branch site protein p14 / SF3b 14 kDa subunit / SF3B14a / Spliceosome-associated protein / ...Pre-mRNA branch site protein p14 / SF3b 14 kDa subunit / SF3B14a / Spliceosome-associated protein / 14-kDa / Splicing factor 3b / subunit 6 / 14kDa


Mass: 14606.900 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9Y3B4

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Protein , 1 types, 1 molecules G

#9: 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, 290 molecules

#11: Chemical
ChemComp-ZN / ZINC ION


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


Mass: 18.015 Da / Num. of mol.: 285 / 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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1A-like U2 snRNPCOMPLEXSpliceosomal 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.#1-#100MULTIPLE SOURCES
2A-like U2 snRNPCOMPLEXSpliceosomal complex A-like U2 snRNP#1-#91NATURAL
3BPS oligoCOMPLEXSpliceosomal complex A-like U2 snRNP#101RECOMBINANT
Molecular weightValue: 1.08 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic construct (others)32630
Source (recombinant)Organism: synthetic construct (others)
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
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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 FIELDBright-field microscopy / 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: 52.03 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 15681
EM imaging opticsEnergyfilter slit width: 20 eV
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
11REFMAC5.8model refinement
12LIBGmodel refinement
13cryoSPARC2.15initial Euler assignmentAb-initio reconstruction
14RELION3.1final Euler assignment
15RELION3.1classification
16RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1470005
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 158286 / Symmetry type: POINT
Atomic model buildingSpace: RECIPROCAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
17Q3L

7q3l

A1
27Q3L

7q3l

B1
37Q3L

7q3l

C1
47Q3L

7q3l

E1
53LQV

3lqv

F112-101
67Q3L

7q3l

G1
77ABH

7abh

1142-85
87Q3L

7q3l

21
97Q3L

7q3l

91
RefinementResolution: 2.2→156.16 Å / Cor.coef. Fo:Fc: 0.904 / SU B: 6.165 / SU ML: 0.143 / ESU R: 0.166
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.33973 --
obs0.33973 515197 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 116.739 Å2
Baniso -1Baniso -2Baniso -3
1--0.01 Å20.65 Å20.09 Å2
2--0.58 Å20.05 Å2
3----0.57 Å2
Refinement stepCycle: 1 / Total: 21218
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0050.01221477
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.4651.66229296
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.5145.2862643
ELECTRON MICROSCOPYr_dihedral_angle_2_deg33.7622.1821077
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.724153525
ELECTRON MICROSCOPYr_dihedral_angle_4_deg20.77615139
ELECTRON MICROSCOPYr_chiral_restr0.1160.2012887
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.0215889
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_it0.95411.88910051
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it1.79817.82612534
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it0.75211.69611426
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined9.41291750
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 -
Rwork0.794 38207 -
obs--100 %

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