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Yorodumi- EMDB-6723: Drosophila full length cryo-EM structure of C3PO complex with a p... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6723 | |||||||||
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Title | Drosophila full length cryo-EM structure of C3PO complex with a point mutation on TRAX subunit at E126 position. | |||||||||
Map data | ||||||||||
Sample |
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Biological species | Drosophila (fruit flies) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 19.7 Å | |||||||||
Authors | Mo X / Yuan AY | |||||||||
Citation | Journal: Nucleic Acids Res / Year: 2018 Title: Structural insights into Drosophila-C3PO complex assembly and 'Dynamic Side Port' model in substrate entry and release. Authors: Xiaobing Mo / Xia Yang / Yuren Adam Yuan / Abstract: In Drosophila and human, component 3 promoter of RISC (C3PO), a heteromeric complex, enhances RISC assembly and promotes RISC activity. Here, we report crystal structure of full-length Drosophila ...In Drosophila and human, component 3 promoter of RISC (C3PO), a heteromeric complex, enhances RISC assembly and promotes RISC activity. Here, we report crystal structure of full-length Drosophila C3PO (E126Q), an inactive C3PO mutant displaying much weaker RNA binding ability, at 2.1 Å resolution. In addition, we also report the cryo-EM structures of full-length Drosophila C3PO (E126Q), C3PO (WT) and SUMO-C3PO (WT, sumo-TRAX + Translin) particles trapped at different conformations at 12, 19.7 and 12.8 Å resolutions, respectively. Crystal structure of C3PO (E126Q) displays a half-barrel architecture consisting of two Trax/Translin heterodimers, whereas cryo-EM structures of C3PO (E126Q), C3PO (WT) and SUMO-C3PO (WT) adopt a closed football-like shape with a hollow interior cavity. Remarkably, both cryo-EM structures of Drosophila C3PO (E126Q) and Drosophila SUMO-C3PO (WT) particles contain a wide side port (∼25 Å × ∼30 Å versus ∼15 Å × ∼20 Å) for RNA substrate entry and release, formed by a pair of anti-parallel packed long α1 helices of TRAX subunits. Notably, cryo-EM structure of SUMO-C3PO showed that four copies of extra densities belonging to N-terminal SUMO tag are located at the outside shell of SUMO-C3PO particle, which demonstrated that the stoichiometry of TRAX/Translin for the in vitro expressed and assembled full-length Drosophila-SUMO-C3PO particle is 4:4, suggesting Drosophila C3PO is composed by TRAX/translin at a ratio of 4:4. Remarkably, the comparison of the cryo-EM structures suggests that the C3PO side ports regulated by α1 helices of TRAX molecules are highly dynamic. Hence, we propose that C3PO particles could adopt a 'Dynamic Side Port' model to capture/digest nucleic acid duplex substrate and release the digested fragments through the dynamic side ports. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6723.map.gz | 1.5 MB | EMDB map data format | |
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Header (meta data) | emd-6723-v30.xml emd-6723.xml | 13.8 KB 13.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_6723_fsc.xml | 5.1 KB | Display | FSC data file |
Images | emd_6723.png | 39 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6723 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6723 | HTTPS FTP |
-Validation report
Summary document | emd_6723_validation.pdf.gz | 78.4 KB | Display | EMDB validaton report |
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Full document | emd_6723_full_validation.pdf.gz | 77.6 KB | Display | |
Data in XML | emd_6723_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6723 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6723 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6723.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 1.69 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : C3PO
Entire | Name: C3PO |
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Components |
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-Supramolecule #1: C3PO
Supramolecule | Name: C3PO / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 Details: In Drosophila, component 3 promoter of RISC (C3PO)is a complex, consisting of 4 Trax/Translin heterodimers. |
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Source (natural) | Organism: Drosophila (fruit flies) |
Recombinant expression | Organism: E.COLi (others) / Recombinant strain: DH5a / Recombinant plasmid: pET-Duet |
Recombinant expression | Organism: E.COLI (others) / Recombinant strain: DH5a / Recombinant plasmid: pET-Duet |
-Supramolecule #2: TRAX_E126Q
Supramolecule | Name: TRAX_E126Q / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 Details: Translin-associated factor X, with a point mutation at E126 position |
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-Supramolecule #3: Translin
Supramolecule | Name: Translin / type: complex / ID: 3 / Parent: 1 / Details: A key component in C3PO complex |
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-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL | |||||||||||||||
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Buffer | pH: 7.4 Component:
Details: 20mM Tris (pH 7.4), 500mM NaCl, 1mM DTT and 2mM EDTA | |||||||||||||||
Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: FORMVAR / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 5 seconds before pluning. | |||||||||||||||
Details | This sample was monodisperse. |
-Electron microscopy
Microscope | JEOL 2200FS |
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Temperature | Min: 70.0 K / Max: 70.0 K |
Details | Preliminary grid screening was performed manually. |
Image recording | Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number grids imaged: 20 / Number real images: 200 / Average exposure time: 1.0 sec. / Average electron dose: 20.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated defocus max: 5.0 µm / Calibrated defocus min: 1.2 µm / Calibrated magnification: 67000 / Illumination mode: OTHER / Imaging mode: DARK FIELD / Cs: 2.2 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 67000 |
Sample stage | Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER Cooling holder cryogen: NITROGEN |
+Image processing
-Atomic model buiding 1
Refinement | Space: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 300 |
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