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- EMDB-1646: Structure of the Human Dicer-TRBP Complex by Electron Microscopy -

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Basic information

Entry
Database: EMDB / ID: EMD-1646
TitleStructure of the Human Dicer-TRBP Complex by Electron Microscopy
Map dataThis is the 3D volume of human Dicer-TRBP complex
Sample
  • Sample: Dicer-TRBP complex
  • Protein or peptide: Human Dicer
  • Protein or peptide: TAR RNA binding protein
KeywordsDicer Enzyme / Ribonuclease III / MicroRNA Processing / Single Particle Electron Microscopy
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsLau PW / Potter CS / Carragher B / MacRae IJ
CitationJournal: Structure / Year: 2009
Title: Structure of the human Dicer-TRBP complex by electron microscopy.
Authors: Pick-Wei Lau / Clinton S Potter / Bridget Carragher / Ian J MacRae /
Abstract: Dicer is a specialized ribonuclease that initiates RNA interference (RNAi) by cleaving double-stranded RNA (dsRNA) into small RNA fragments about 22 nucleotides long. Here, we present the three- ...Dicer is a specialized ribonuclease that initiates RNA interference (RNAi) by cleaving double-stranded RNA (dsRNA) into small RNA fragments about 22 nucleotides long. Here, we present the three-dimensional structure of human Dicer bound to the protein TRBP at approximately 20 A resolution determined by negative-stain electron microscopy (EM) and single-particle analysis. Our analysis reveals that the Dicer-TRBP complex is an L-shaped molecule with a long edge of 150 A and a 100 A extension on one end. A surface trench runs the length of the long edge of the molecule, defining a putative dsRNA-binding site. Docking the crystal structure of Giardia Dicer, which represents the nuclease core of human Dicer, into the EM map suggests two possible overall molecular architectures for human Dicer. These results offer insights into the structure of Dicer proteins found in multicellular organisms and provide a conceptual framework for understanding the initiation of RNAi.
History
DepositionAug 22, 2009-
Header (metadata) releaseAug 28, 2009-
Map releaseOct 20, 2009-
UpdateOct 20, 2009-
Current statusOct 20, 2009Processing site: PDBe / 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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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1646_ima...

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Map

FileDownload / File: emd_1646.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the 3D volume of human Dicer-TRBP complex
Voxel sizeX=Y=Z: 3.093 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-1.03642 - 3.11741
Average (Standard dev.)-0.000000000002081 (±0.149105)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-60-60-60
Dimensions120120120
Spacing120120120
CellA=B=C: 371.16 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.0933.0933.093
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z371.160371.160371.160
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-60-60-60
NC/NR/NS120120120
D min/max/mean-1.0363.117-0.000

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Supplemental data

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Sample components

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Entire : Dicer-TRBP complex

EntireName: Dicer-TRBP complex
Components
  • Sample: Dicer-TRBP complex
  • Protein or peptide: Human Dicer
  • Protein or peptide: TAR RNA binding protein

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Supramolecule #1000: Dicer-TRBP complex

SupramoleculeName: Dicer-TRBP complex / type: sample / ID: 1000 / Details: The sample was mostly monodisperse
Oligomeric state: One human Dicer binds to one TRBP molecule
Number unique components: 2
Molecular weightTheoretical: 250 KDa

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Macromolecule #1: Human Dicer

MacromoleculeName: Human Dicer / type: protein_or_peptide / ID: 1 / Name.synonym: hDicer / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 220 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: Baculovirus

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Macromolecule #2: TAR RNA binding protein

MacromoleculeName: TAR RNA binding protein / type: protein_or_peptide / ID: 2 / Name.synonym: TRBP / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 30 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: Baculovirus

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: 150mM KCl, 20mM HEPES
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 2% w/v uranyl acetate for approximately 20 seconds.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus min: 0.15 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Single tilt room temperature grid holder / Specimen holder model: SIDE ENTRY, EUCENTRIC
DateMay 31, 2009
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Number real images: 947 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Whole image
Final two d classificationNumber classes: 711
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: OTHER / Software - Name: EMAN, SPIDER / Number images used: 129554

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Atomic model buiding 1

Initial modelPDB ID:

2ffl


Chain - Chain ID: A
SoftwareName: Chimera
DetailsPDBEntryID_givenInChain. Protocol: Rigid Body. Manual docking
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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