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- EMDB-6899: Full length cryo-EM structure of Drosophila-sumo-C3PO (sumo-TRAX ... -

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Entry
Database: EMDB / ID: EMD-6899
TitleFull length cryo-EM structure of Drosophila-sumo-C3PO (sumo-TRAX + translin) complex
Map dataDrosophila full length cryoEM structure of sumo-C3PO complex (sumo-TRAX translin)
Sample
  • Complex: C3PO
    • Complex: sumo-TRAX
    • Complex: Translin
Biological speciesDrosophila (fruit flies)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.8 Å
AuthorsMo X / Yuan AY
CitationJournal: 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
DepositionJan 22, 2018-
Header (metadata) releaseFeb 7, 2018-
Map releaseFeb 7, 2018-
UpdateOct 3, 2018-
Current statusOct 3, 2018Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6899.png

Downloads & links

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Map

FileDownload / File: emd_6899.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDrosophila full length cryoEM structure of sumo-C3PO complex (sumo-TRAX translin)
Voxel sizeX=Y=Z: 1.69 Å
Density
Contour LevelBy AUTHOR: 1. / Movie #1: 1
Minimum - Maximum-2.8151772 - 6.9517846
Average (Standard dev.)0.0664688 (±0.52904373)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-75-75-75
Dimensions150150150
Spacing150150150
CellA=B=C: 253.50002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.691.691.69
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z253.500253.500253.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-75-75-75
NC/NR/NS150150150
D min/max/mean-2.8156.9520.066

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Supplemental data

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Sample components

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Entire : C3PO

EntireName: C3PO
Components
  • Complex: C3PO
    • Complex: sumo-TRAX
    • Complex: Translin

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Supramolecule #1: C3PO

SupramoleculeName: C3PO / type: complex / ID: 1 / Parent: 0
Details: In Drosophila, component 3 promoter of RISC (C3PO)is a complex, consisting of 4 Trax/Translin heterodimers.
Source (natural)Organism: Drosophila (fruit flies)
Recombinant expressionOrganism: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
Recombinant strain: DH5a / Recombinant plasmid: pET-Duet
Recombinant expressionOrganism: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
Recombinant strain: BL21 (DE3) / Recombinant plasmid: pET-Duet
Molecular weightExperimental: 320 KDa

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Supramolecule #2: sumo-TRAX

SupramoleculeName: sumo-TRAX / type: complex / ID: 2 / Parent: 1 / Details: Translin-associated factor X

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Supramolecule #3: Translin

SupramoleculeName: Translin / type: complex / ID: 3 / Parent: 1 / Details: A key component in C3PO complex

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
0.5 mMNaClSodium chloridesodium chloride
20.0 mMTrisTris
2.0 mMDTTDTT
1.0 mMEDTAEthylenediaminetetraacetic acidEDTAEthylenediaminetetraacetic acid

Details: 20mM Tris (pH 7.4), 500mM NaCl, 1mM DTT and 2mM EDTA
GridModel: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 5 seconds before pluning.
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated defocus max: 5.0 µm / Calibrated defocus min: 1.2 µm / Calibrated magnification: 47000 / Illumination mode: OTHER / Imaging mode: DARK FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 47000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
DetailsPreliminary grid screening was performed manually.
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number grids imaged: 20 / Number real images: 800 / Average exposure time: 1.0 sec. / Average electron dose: 25.0 e/Å2

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Image processing

Particle selectionNumber selected: 24000
CTF correctionSoftware - Name: EMAN2 (ver. 2.1)
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 32 / Avg.num./class: 200 / Software - Name: EMAN2 (ver. 2.1)
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 128 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 12.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 (ver. 2.1) / Number images used: 13200
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 200

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