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Yorodumi- PDB-7px3: Structure of U5 snRNP assembly and recycling factor TSSC4 in comp... -
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-Basic information
Entry | Database: PDB / ID: 7px3 | ||||||
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Title | Structure of U5 snRNP assembly and recycling factor TSSC4 in complex with BRR2 and Jab1 domain of PRPF8 | ||||||
Components |
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Keywords | SPLICING / Spliceosomal biogenesis | ||||||
Function / homology | Function and homology information cis assembly of pre-catalytic spliceosome / molecular sequestering activity / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / U2-type catalytic step 1 spliceosome / RNA splicing, via transesterification reactions / U2-type precatalytic spliceosome / U2-type catalytic step 2 spliceosome / K63-linked polyubiquitin modification-dependent protein binding / mRNA Splicing - Minor Pathway / spliceosomal tri-snRNP complex assembly ...cis assembly of pre-catalytic spliceosome / molecular sequestering activity / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / U2-type catalytic step 1 spliceosome / RNA splicing, via transesterification reactions / U2-type precatalytic spliceosome / U2-type catalytic step 2 spliceosome / K63-linked polyubiquitin modification-dependent protein binding / mRNA Splicing - Minor Pathway / spliceosomal tri-snRNP complex assembly / U5 snRNA binding / U5 snRNP / U2 snRNA binding / U6 snRNA binding / spliceosomal snRNP assembly / pre-mRNA intronic binding / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / RNA splicing / helicase activity / spliceosomal complex / mRNA processing / mRNA splicing, via spliceosome / osteoblast differentiation / cellular response to tumor necrosis factor / cellular response to lipopolysaccharide / RNA helicase activity / RNA helicase / nuclear speck / protein-containing complex binding / ATP hydrolysis activity / RNA binding / nucleoplasm / ATP binding / identical protein binding / membrane / nucleus / cytoplasm Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.05 Å | ||||||
Authors | Bergfort, A. / Kuropka, B. / Ilik, I.A. / Freund, C. / Aktas, T. / Hilal, T. / Weber, G. / Wahl, M.C. | ||||||
Funding support | Germany, 1items
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Citation | Journal: Nucleic Acids Res / Year: 2022 Title: The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling. Authors: Alexandra Bergfort / Tarek Hilal / Benno Kuropka / İbrahim Avşar Ilik / Gert Weber / Tuğçe Aktaş / Christian Freund / Markus C Wahl / Abstract: Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. ...Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. The intrinsically disordered TSSC4 protein has been identified as a nuclear-localized U5 snRNP and U4/U6-U5 tri-snRNP assembly/recycling factor, but how TSSC4's intrinsic disorder supports TSSC4 functions remains unknown. Using diverse interaction assays and cryogenic electron microscopy-based structural analysis, we show that TSSC4 employs four conserved, non-contiguous regions to bind the PRPF8 Jab1/MPN domain and the SNRNP200 helicase at functionally important sites. It thereby inhibits SNRNP200 helicase activity, spatially aligns the proteins, coordinates formation of a U5 sub-module and transiently blocks premature interaction of SNRNP200 with at least three other spliceosomal factors. Guided by the structure, we designed a TSSC4 variant that lacks stable binding to the PRPF8 Jab1/MPN domain or SNRNP200 in vitro. Comparative immunoprecipitation/mass spectrometry from HEK293 nuclear extract revealed distinct interaction profiles of wild type TSSC4 and the variant deficient in PRPF8/SNRNP200 binding with snRNP proteins, other spliceosomal proteins as well as snRNP assembly/recycling factors and chaperones. Our findings elucidate molecular strategies employed by an intrinsically disordered protein to promote snRNP assembly, and suggest multiple TSSC4-dependent stages during snRNP assembly/recycling. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7px3.cif.gz | 389.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7px3.ent.gz | 297.4 KB | Display | PDB format |
PDBx/mmJSON format | 7px3.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7px3_validation.pdf.gz | 789.4 KB | Display | wwPDB validaton report |
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Full document | 7px3_full_validation.pdf.gz | 812.6 KB | Display | |
Data in XML | 7px3_validation.xml.gz | 60.3 KB | Display | |
Data in CIF | 7px3_validation.cif.gz | 91.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/px/7px3 ftp://data.pdbj.org/pub/pdb/validation_reports/px/7px3 | HTTPS FTP |
-Related structure data
Related structure data | 13690MC 7os1C 7os2C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 199666.656 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: SNRNP200, ASCC3L1, HELIC2, KIAA0788 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O75643, RNA helicase |
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#2: Protein | Mass: 30133.229 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: PRPF8, PRPC8 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q6P2Q9 |
#3: Protein | Mass: 34366.465 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: TSSC4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9Y5U2 |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Buffer solution | pH: 7.6 | ||||||||||||||||||||||||||||||
Specimen | Conc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 308 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 120000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 30.59 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3349 |
-Processing
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1025529 | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 387973 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 115.8 Å2 | ||||||||||||||||||||||||
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