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- EMDB-13028: The U2 part of Saccharomyces cerevisiae spliceosomal pre-A comple... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-13028 | |||||||||
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Title | The U2 part of Saccharomyces cerevisiae spliceosomal pre-A complex (delta BS-A ACT1) | |||||||||
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![]() | S. cerevisiae / pre-A complex / Prp5 / U1 snRNP / U2 snRNP / prespliceosome / SPLICING | |||||||||
Function / homology | ![]() mRNA branch site recognition / splicing factor binding / U4/U6 snRNP / 7-methylguanosine cap hypermethylation / pICln-Sm protein complex / ATP-dependent activity, acting on RNA / small nuclear ribonucleoprotein complex / SMN-Sm protein complex / spliceosomal tri-snRNP complex / poly(U) RNA binding ...mRNA branch site recognition / splicing factor binding / U4/U6 snRNP / 7-methylguanosine cap hypermethylation / pICln-Sm protein complex / ATP-dependent activity, acting on RNA / small nuclear ribonucleoprotein complex / SMN-Sm protein complex / spliceosomal tri-snRNP complex / poly(U) RNA binding / U2-type spliceosomal complex / mRNA cis splicing, via spliceosome / commitment complex / U2-type prespliceosome assembly / U4 snRNP / U2 snRNP / positive regulation of mRNA splicing, via spliceosome / U1 snRNP / U2-type prespliceosome / precatalytic spliceosome / spliceosomal complex assembly / regulation of alternative mRNA splicing, via spliceosome / mRNA 5'-splice site recognition / regulation of RNA splicing / U5 snRNP / U2 snRNA binding / spliceosomal snRNP assembly / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / spliceosomal complex / mRNA splicing, via spliceosome / nucleic acid binding / RNA helicase activity / RNA helicase / response to xenobiotic stimulus / mRNA binding / nucleolus / ATP hydrolysis activity / RNA binding / zinc ion binding / ATP binding / nucleus / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 9.0 Å | |||||||||
![]() | Zhang Z / Rigo N | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Structural insights into how Prp5 proofreads the pre-mRNA branch site. Authors: Zhenwei Zhang / Norbert Rigo / Olexandr Dybkov / Jean-Baptiste Fourmann / Cindy L Will / Vinay Kumar / Henning Urlaub / Holger Stark / Reinhard Lührmann / ![]() Abstract: During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly ...During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. ). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2-BS helix), which is proofread by Prp5 at this stage through an unclear mechanism. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2-BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155 to the bulged BS-A of the U2-BS helix triggers closure of Hsh155, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 37.6 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 39.2 KB 39.2 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 5.1 KB | Display | ![]() |
Images | ![]() | 82.8 KB | ||
Filedesc metadata | ![]() | 10.8 KB | ||
Others | ![]() ![]() | 28.2 MB 28.3 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 663.8 KB | Display | ![]() |
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Full document | ![]() | 663.4 KB | Display | |
Data in XML | ![]() | 13.5 KB | Display | |
Data in CIF | ![]() | 16.9 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7oqbMC ![]() 7oqcC ![]() 7oqeC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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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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Voxel size | X=Y=Z: 2.32 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: #2
File | emd_13028_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_13028_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
+Entire : S. cerevisiae spliceosomal pre-A complex
+Supramolecule #1: S. cerevisiae spliceosomal pre-A complex
+Macromolecule #1: U2 snRNP component HSH155
+Macromolecule #3: Pre-mRNA-splicing factor PRP11
+Macromolecule #4: Pre-mRNA-splicing factor PRP21
+Macromolecule #5: Pre-mRNA-splicing factor PRP9
+Macromolecule #6: Pre-mRNA-splicing factor RDS3
+Macromolecule #7: Cold sensitive U2 snRNA suppressor 1
+Macromolecule #8: Pre-mRNA-splicing factor RSE1
+Macromolecule #9: Protein HSH49
+Macromolecule #10: RDS3 complex subunit 10
+Macromolecule #11: U2 small nuclear ribonucleoprotein A'
+Macromolecule #12: U2 small nuclear ribonucleoprotein B''
+Macromolecule #13: Small nuclear ribonucleoprotein-associated protein B
+Macromolecule #14: Small nuclear ribonucleoprotein Sm D1
+Macromolecule #15: Small nuclear ribonucleoprotein Sm D2
+Macromolecule #16: Small nuclear ribonucleoprotein Sm D3
+Macromolecule #17: Small nuclear ribonucleoprotein E
+Macromolecule #18: Small nuclear ribonucleoprotein F
+Macromolecule #19: Small nuclear ribonucleoprotein G
+Macromolecule #20: Pre-mRNA-processing ATP-dependent RNA helicase PRP5
+Macromolecule #2: ACT1 pre-mRNA (delta-BS-A)
+Macromolecule #21: U2 snRNA
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.9 |
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Grid | Model: Quantifoil R3.5/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Average exposure time: 1.02 sec. / Average electron dose: 44.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |