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- EMDB-4686: Human U5.U4/U6 tri-snRNP, refined after focused classification on... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-4686 | |||||||||
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Title | Human U5.U4/U6 tri-snRNP, refined after focused classification on Prp28 | |||||||||
![]() | Human U5.U4/U6 tri-snRNP, refined after focused classification on Prp28 | |||||||||
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Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.98 Å | |||||||||
![]() | Charenton C / Wilkinson ME / Nagai K | |||||||||
![]() | ![]() Title: Mechanism of 5' splice site transfer for human spliceosome activation. Authors: Clément Charenton / Max E Wilkinson / Kiyoshi Nagai / ![]() Abstract: The prespliceosome, comprising U1 and U2 small nuclear ribonucleoproteins (snRNPs) bound to the precursor messenger RNA 5' splice site (5'SS) and branch point sequence, associates with the U4/U6.U5 ...The prespliceosome, comprising U1 and U2 small nuclear ribonucleoproteins (snRNPs) bound to the precursor messenger RNA 5' splice site (5'SS) and branch point sequence, associates with the U4/U6.U5 tri-snRNP to form the fully assembled precatalytic pre-B spliceosome. Here, we report cryo-electron microscopy structures of the human pre-B complex captured before U1 snRNP dissociation at 3.3-angstrom core resolution and the human tri-snRNP at 2.9-angstrom resolution. U1 snRNP inserts the 5'SS-U1 snRNA helix between the two RecA domains of the Prp28 DEAD-box helicase. Adenosine 5'-triphosphate-dependent closure of the Prp28 RecA domains releases the 5'SS to pair with the nearby U6 ACAGAGA-box sequence presented as a mobile loop. The structures suggest that formation of the 5'SS-ACAGAGA helix triggers remodeling of an intricate protein-RNA network to induce Brr2 helicase relocation to its loading sequence in U4 snRNA, enabling Brr2 to unwind the U4/U6 snRNA duplex to allow U6 snRNA to form the catalytic center of the spliceosome. | |||||||||
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 | ![]() | 260.1 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 15.2 KB 15.2 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 14.9 KB | Display | ![]() |
Images | ![]() | 204.2 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 4658C ![]() 4665C ![]() 4672C ![]() 4673C ![]() 4674C ![]() 4675C ![]() 4676C ![]() 4687C ![]() 4688C ![]() 4689C ![]() 4690C ![]() 6qw6C ![]() 6qx9C C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() Data #1: Dataset 1 of human pre-B spliceosome; motion-corrected micrographs [micrographs - single frame] Data #2: Dataset 2 of human pre-B spliceosome; motion-corrected micrographs [micrographs - single frame] Data #3: Dataset 3 of human pre-B spliceosome; motion-corrected micrographs [micrographs - single frame] Data #4: Dataset 4 of human pre-B spliceosome; motion-corrected micrographs [micrographs - single frame] Data #5: Dataset 5 of human pre-B spliceosome; motion-corrected micrographs [micrographs - single frame] Data #6: Selected U4/U6.U5 tri-snRNP particles after Bayesian polishing [picked particles - single frame - processed] Data #7: Crude shifted preB particles [picked particles - single frame - unprocessed]) |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Human U5.U4/U6 tri-snRNP, refined after focused classification on Prp28 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.022 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Human U4/U6.U5 tri-snRNP
Entire | Name: Human U4/U6.U5 tri-snRNP |
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Components |
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-Supramolecule #1: Human U4/U6.U5 tri-snRNP
Supramolecule | Name: Human U4/U6.U5 tri-snRNP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#32 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 1.7 MDa |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 1.3 mg/mL | ||||||||
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Buffer | pH: 7.9 Component:
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Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III / Details: Wait 30s, blot for 2s to 3s.. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Average exposure time: 6.0 sec. / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / 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 |