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- EMDB-14146: Structural organization of a late activated human spliceosome (Ba... -
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Basic information
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Title | Structural organization of a late activated human spliceosome (Baqr, core region) | |||||||||
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![]() | Spliceosome / helicase / Prp2 / Aquarius / catalytic activation / SPLICING | |||||||||
Function / homology | ![]() RES complex / negative regulation of chemokine-mediated signaling pathway / snoRNA splicing / U11/U12 snRNP / post-mRNA release spliceosomal complex / regulation of retinoic acid receptor signaling pathway / U2 snRNP binding / U7 snRNA binding / histone pre-mRNA DCP binding / U7 snRNP ...RES complex / negative regulation of chemokine-mediated signaling pathway / snoRNA splicing / U11/U12 snRNP / post-mRNA release spliceosomal complex / regulation of retinoic acid receptor signaling pathway / U2 snRNP binding / U7 snRNA binding / histone pre-mRNA DCP binding / U7 snRNP / B-WICH complex / regulation of vitamin D receptor signaling pathway / histone pre-mRNA 3'end processing complex / SLBP independent Processing of Histone Pre-mRNAs / SLBP Dependent Processing of Replication-Dependent Histone Pre-mRNAs / embryonic brain development / splicing factor binding / protein methylation / U12-type spliceosomal complex / methylosome / nuclear retinoic acid receptor binding / Prp19 complex / 7-methylguanosine cap hypermethylation / positive regulation of androgen receptor activity / U1 snRNP binding / pICln-Sm protein complex / pre-mRNA binding / U2-type catalytic step 1 spliceosome / RNA splicing, via transesterification reactions / small nuclear ribonucleoprotein complex / SMN-Sm protein complex / C2H2 zinc finger domain binding / regulation of mRNA splicing, via spliceosome / spliceosomal tri-snRNP complex / P granule / telomerase holoenzyme complex / U2-type spliceosomal complex / mRNA cis splicing, via spliceosome / positive regulation by host of viral transcription / U2-type precatalytic spliceosome / commitment complex / positive regulation of vitamin D receptor signaling pathway / telomerase RNA binding / U2-type prespliceosome assembly / nuclear vitamin D receptor binding / U2-type catalytic step 2 spliceosome / U4 snRNP / Notch binding / Regulation of gene expression in late stage (branching morphogenesis) pancreatic bud precursor cells / RUNX3 regulates NOTCH signaling / U2 snRNP / SAGA complex / positive regulation of mRNA splicing, via spliceosome / RNA Polymerase II Transcription Termination / NOTCH4 Intracellular Domain Regulates Transcription / positive regulation of transcription by RNA polymerase III / Basigin interactions / U1 snRNP / ubiquitin-ubiquitin ligase activity / WD40-repeat domain binding / NOTCH3 Intracellular Domain Regulates Transcription / pattern recognition receptor activity / positive regulation of neurogenesis / U2-type prespliceosome / K63-linked polyubiquitin modification-dependent protein binding / cyclosporin A binding / nuclear androgen receptor binding / positive regulation of transcription by RNA polymerase I / precatalytic spliceosome / Notch-HLH transcription pathway / positive regulation of transforming growth factor beta receptor signaling pathway / Formation of paraxial mesoderm / spliceosomal complex assembly / mRNA Splicing - Minor Pathway / SMAD binding / regulation of RNA splicing / antiviral innate immune response / mRNA 3'-splice site recognition / protein localization to nucleus / spliceosomal tri-snRNP complex assembly / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / retinoic acid receptor signaling pathway / U5 snRNA binding / positive regulation of G1/S transition of mitotic cell cycle / U5 snRNP / U2 snRNA binding / U6 snRNA binding / regulation of DNA repair / spliceosomal snRNP assembly / protein peptidyl-prolyl isomerization / Cajal body / pre-mRNA intronic binding / cellular response to retinoic acid / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / RNA splicing / helicase activity / positive regulation of RNA splicing Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
![]() | Cretu C / Pena V | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Structural basis of catalytic activation in human splicing. Authors: Jana Schmitzová / Constantin Cretu / Christian Dienemann / Henning Urlaub / Vladimir Pena / ![]() ![]() Abstract: Pre-mRNA splicing follows a pathway driven by ATP-dependent RNA helicases. A crucial event of the splicing pathway is the catalytic activation, which takes place at the transition between the ...Pre-mRNA splicing follows a pathway driven by ATP-dependent RNA helicases. A crucial event of the splicing pathway is the catalytic activation, which takes place at the transition between the activated B and the branching-competent B spliceosomes. Catalytic activation occurs through an ATP-dependent remodelling mediated by the helicase PRP2 (also known as DHX16). However, because PRP2 is observed only at the periphery of spliceosomes, its function has remained elusive. Here we show that catalytic activation occurs in two ATP-dependent stages driven by two helicases: PRP2 and Aquarius. The role of Aquarius in splicing has been enigmatic. Here the inactivation of Aquarius leads to the stalling of a spliceosome intermediate-the B complex-found halfway through the catalytic activation process. The cryogenic electron microscopy structure of B reveals how PRP2 and Aquarius remodel B and B, respectively. Notably, PRP2 translocates along the intron while it strips away the RES complex, opens the SF3B1 clamp and unfastens the branch helix. Translocation terminates six nucleotides downstream of the branch site through an assembly of PPIL4, SKIP and the amino-terminal domain of PRP2. Finally, Aquarius enables the dissociation of PRP2, plus the SF3A and SF3B complexes, which promotes the relocation of the branch duplex for catalysis. This work elucidates catalytic activation in human splicing, reveals how a DEAH helicase operates and provides a paradigm for how helicases can coordinate their activities. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 427 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 71.8 KB 71.8 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 18.3 KB | Display | ![]() |
Images | ![]() | 40.4 KB | ||
Masks | ![]() | 536.4 MB | ![]() | |
Filedesc metadata | ![]() | 21.9 KB | ||
Others | ![]() ![]() | 428.6 MB 428 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 812.5 KB | Display | ![]() |
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Full document | ![]() | 812.1 KB | Display | |
Data in XML | ![]() | 26.4 KB | Display | |
Data in CIF | ![]() | 35.2 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7qttMC ![]() 8ch6C M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.05 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
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Density Histograms |
-Half map: #2
File | emd_14146_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
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Density Histograms |
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Sample components
+Entire : Late human activated spliceosome arrested with a dominant-negativ...
+Supramolecule #1: Late human activated spliceosome arrested with a dominant-negativ...
+Macromolecule #1: Splicing factor 3B subunit 3
+Macromolecule #2: Splicing factor 3B subunit 5
+Macromolecule #3: Splicing factor 3B subunit 1
+Macromolecule #4: PHD finger-like domain-containing protein 5A
+Macromolecule #5: Splicing factor 3B subunit 2
+Macromolecule #6: Splicing factor 3B subunit 4
+Macromolecule #7: G-patch domain and KOW motifs-containing protein
+Macromolecule #8: Splicing factor 3A subunit 2
+Macromolecule #9: Splicing factor 3A subunit 3
+Macromolecule #10: BUD13 homolog
+Macromolecule #11: Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX16
+Macromolecule #12: Pleiotropic regulator 1
+Macromolecule #13: Cell division cycle 5-like protein
+Macromolecule #14: Spliceosome-associated protein CWC15 homolog
+Macromolecule #15: Protein BUD31 homolog
+Macromolecule #16: Pre-mRNA-splicing factor RBM22
+Macromolecule #17: Peptidyl-prolyl cis-trans isomerase-like 4
+Macromolecule #18: Pre-mRNA-splicing factor CWC22 homolog
+Macromolecule #19: Crooked neck-like protein 1
+Macromolecule #20: SNW domain-containing protein 1
+Macromolecule #21: RING finger protein 113A
+Macromolecule #22: Pre-mRNA-processing-splicing factor 8
+Macromolecule #23: 116 kDa U5 small nuclear ribonucleoprotein component
+Macromolecule #28: Small nuclear ribonucleoprotein F
+Macromolecule #29: Small nuclear ribonucleoprotein E
+Macromolecule #30: Small nuclear ribonucleoprotein Sm D3
+Macromolecule #31: Small nuclear ribonucleoprotein Sm D1
+Macromolecule #32: Small nuclear ribonucleoprotein Sm D2
+Macromolecule #33: Small nuclear ribonucleoprotein G
+Macromolecule #34: Small nuclear ribonucleoprotein-associated proteins B and B'
+Macromolecule #35: Peptidyl-prolyl cis-trans isomerase-like 1
+Macromolecule #36: Serine/arginine repetitive matrix protein 2
+Macromolecule #37: RING-type E3 ubiquitin-protein ligase PPIL2
+Macromolecule #24: U6snRNA
+Macromolecule #25: U5 snRNA
+Macromolecule #26: U2snRNA
+Macromolecule #27: MINX
+Macromolecule #38: ZINC ION
+Macromolecule #39: INOSITOL HEXAKISPHOSPHATE
+Macromolecule #40: GUANOSINE-5'-TRIPHOSPHATE
+Macromolecule #41: MAGNESIUM ION
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.6 mg/mL | |||||||||||||||
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Buffer | pH: 7.9 Component:
Details: All solutions were sterile filtered using a 0.22um vacuum filtration unit. | |||||||||||||||
Grid | Model: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV Details: Volumes of 4 ul of the concentrated sample were applied to one side of glow-discharged UltrAuFoil 200 2/2 grids (Quantifoil) in a Vitrobot Mark IV (FEI) operating at 4 degrees Celsius and ...Details: Volumes of 4 ul of the concentrated sample were applied to one side of glow-discharged UltrAuFoil 200 2/2 grids (Quantifoil) in a Vitrobot Mark IV (FEI) operating at 4 degrees Celsius and 100% humidity. The grids were blotted for 2s with blotting force 5 and immediately frozen by plunging into liquid ethane.. | |||||||||||||||
Details | The Baqr spliceosome was purified by affinity selection and gradient ultracentrifugation and crosslinked with 0.1% (v/v) glutaraldehyde in batch for cryo-EM grid preparation. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 30 eV |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 10013 / Average exposure time: 9.0 sec. / Average electron dose: 45.47 e/Å2 Details: Automated data acquisition for dataset 1 (untilted, 5229 micrographs) and dataset 2 (tilted, 25 degrees, 4784 micrographs) was performed with FEI EPU software package at a nominal ...Details: Automated data acquisition for dataset 1 (untilted, 5229 micrographs) and dataset 2 (tilted, 25 degrees, 4784 micrographs) was performed with FEI EPU software package at a nominal magnification of 130,000 (1.05 A per pixel). Micrographs for these two datasets, dose fractionated over 40 frames, were collected at a dose rate of 5.04 or 5.06 e/A2/s-1 over 9 s, resulting in a total dose of 45.38 and 45.55 e/A2, respectively. |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Initial model |
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Refinement | Space: REAL / Protocol: OTHER / Overall B value: 177.21 | ||||||||||
Output model | ![]() PDB-7qtt: |