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- EMDB-2216: DOLORS: Versatile Strategy for Internal Labeling and Domain Local... -

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Basic information

Entry
Database: EMDB / ID: EMD-2216
TitleDOLORS: Versatile Strategy for Internal Labeling and Domain Localization in Electron Microscopy
Map dataRNaseIIIb-labeled Dicer
Sample
  • Sample: Dicer labeled with Streptavidin at the RNase IIIb domain
  • Protein or peptide: Human Dicer
  • Protein or peptide: Streptavidin
KeywordsDicer Enzyme / Ribonuclease III / MicroRNA Processing / Single Particle Electron Microscopy
Biological speciesHomo sapiens (human) / Streptomyces avidinii (bacteria)
Methodsingle particle reconstruction / negative staining
AuthorsLau PW / Potter CS / Carragher B / MacRae IJ
CitationJournal: Structure / Year: 2012
Title: DOLORS: versatile strategy for internal labeling and domain localization in electron microscopy.
Authors: Pick-Wei Lau / Clinton S Potter / Bridget Carragher / Ian J MacRae /
Abstract: Single-particle electron microscopy (EM) is a powerful tool for studying the structures of large biological molecules. However, the achievable resolution does not always allow for direct recognition ...Single-particle electron microscopy (EM) is a powerful tool for studying the structures of large biological molecules. However, the achievable resolution does not always allow for direct recognition of individual protein domains. Labels that can be visualized by EM have been developed for protein termini, but tagging internal domains remains a challenge. We describe a robust strategy for determining the position of internal sites within EM maps, termed domain localization by RCT sampling (DOLORS). DOLORS uses monovalent streptavidin added posttranslationally to tagged sites in the target protein. Internal labels generally display less conformational flexibility than terminal labels, providing more precise positional information. Automated methods are used to rapidly generate assemblies of unique 3D models allowing the attachment sites of labeled domains to be accurately identified and thus provide an overall architectural map of the molecule.
History
DepositionOct 11, 2012-
Header (metadata) releaseNov 14, 2012-
Map releaseDec 19, 2012-
UpdateDec 19, 2012-
Current statusDec 19, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image2216.png

Downloads & links

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Map

FileDownload / File: emd_2216.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRNaseIIIb-labeled Dicer
Voxel sizeX=Y=Z: 3.52 Å
Density
Contour LevelBy AUTHOR: 4.5 / Movie #1: 4.5
Minimum - Maximum-4.08396959 - 15.43524742
Average (Standard dev.)-0.04004382 (±0.98056817)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-12-12-12
Dimensions969696
Spacing969696
CellA=B=C: 337.91998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.523.523.52
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z337.920337.920337.920
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-12-12-12
NC/NR/NS969696
D min/max/mean-4.08415.435-0.040

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Supplemental data

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Sample components

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Entire : Dicer labeled with Streptavidin at the RNase IIIb domain

EntireName: Dicer labeled with Streptavidin at the RNase IIIb domain
Components
  • Sample: Dicer labeled with Streptavidin at the RNase IIIb domain
  • Protein or peptide: Human Dicer
  • Protein or peptide: Streptavidin

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Supramolecule #1000: Dicer labeled with Streptavidin at the RNase IIIb domain

SupramoleculeName: Dicer labeled with Streptavidin at the RNase IIIb domain
type: sample / ID: 1000 / Details: The sample was mostly monodisperse
Oligomeric state: One human Dicer and one streptavidin molecule
Number unique components: 2
Molecular weightTheoretical: 280 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)

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Macromolecule #2: Streptavidin

MacromoleculeName: Streptavidin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: Tetramer / Recombinant expression: Yes
Source (natural)Organism: Streptomyces avidinii (bacteria)
Molecular weightTheoretical: 60 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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: 7.5 / Details: 150mM KCl, 25mM HEPES
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 2% w/v uranyl acetate
GridDetails: holey C-flat grids covered with an additional layer of thin carbon
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 / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 50
DateJan 10, 2011
Image recordingNumber real images: 880 / Average electron dose: 20 e/Å2
Tilt angle min0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final two d classificationNumber classes: 1
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Software - Name: Spider / Number images used: 27607
DetailsProcessing was done using APPION pipeline

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Atomic model buiding 1

Initial modelPDB ID:

2ffl


Chain - Chain ID: A
SoftwareName: Chimera
DetailsProtocol: Rigid body. manual docking based upon overall fit and correspondence to tagging data
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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