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- EMDB-9266: Single-Molecule 3D Image of DNA Origami Bennett Linkage by Image ... -

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Basic information

Entry
Database: EMDB / ID: EMD-9266
TitleSingle-Molecule 3D Image of DNA Origami Bennett Linkage by Image Contrast Enhancement and Individual Particle Electron Tomography (No. 01)
Map dataOrigami Bennett Linkage (No. 01)
Sample
  • Complex: DNA origami Bennett linkage
    • Complex: M13 phage genome segment
    • Complex: Synthetic DNA oligonucleotides
Biological speciesEnterobacteria phage M13 (virus) / synthetic construct (others)
Methodelectron tomography / cryo EM / negative staining / Resolution: 97.7 Å
AuthorsWu H / Zhai X / Lei D / Liu J / Yu Y / Bie R / Ren G
CitationJournal: Sci Rep / Year: 2018
Title: An Algorithm for Enhancing the Image Contrast of Electron Tomography.
Authors: Hao Wu / Xiaobo Zhai / Dongsheng Lei / Jianfang Liu / Yadong Yu / Rongfang Bie / Gang Ren /
Abstract: Three-dimensional (3D) reconstruction of a single protein molecule is essential for understanding the relationship between the structural dynamics and functions of the protein. Electron tomography ...Three-dimensional (3D) reconstruction of a single protein molecule is essential for understanding the relationship between the structural dynamics and functions of the protein. Electron tomography (ET) provides a tool for imaging an individual particle of protein from a series of tilted angles. Individual-particle electron tomography (IPET) provides an approach for reconstructing a 3D density map from a single targeted protein particle (without averaging from different particles of this type of protein), in which the target particle was imaged from a series of tilting angles. However, owing to radiation damage limitations, low-dose images (high noise, and low image contrast) are often challenging to be aligned for 3D reconstruction at intermediate resolution (1-3 nm). Here, we propose a computational method to enhance the image contrast, without increasing any experimental dose, for IPET 3D reconstruction. Using an edge-preserving smoothing-based multi-scale image decomposition algorithm, this method can detect the object against a high-noise background and enhance the object image contrast without increasing the noise level or significantly decreasing the image resolution. The method was validated by using both negative staining (NS) ET and cryo-ET images. The successful 3D reconstruction of a small molecule (<100 kDa) indicated that this method can be used as a supporting tool to current ET 3D reconstruction methods for studying protein dynamics via structure determination from each individual particle of the same type of protein.
History
DepositionOct 26, 2018-
Header (metadata) releaseNov 28, 2018-
Map releaseDec 12, 2018-
UpdateDec 12, 2018-
Current statusDec 12, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9266.png

Downloads & links

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Map

FileDownload / File: emd_9266.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOrigami Bennett Linkage (No. 01)
Voxel sizeX=Y=Z: 3.7 Å
Density
Contour LevelMovie #1: 0.25
Minimum - Maximum-0.34884658 - 1.0290926
Average (Standard dev.)0.006440866 (±0.06782825)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 947.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.73.73.7
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z947.200947.200947.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-0.3491.0290.006

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Supplemental data

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Sample components

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Entire : DNA origami Bennett linkage

EntireName: DNA origami Bennett linkage
Components
  • Complex: DNA origami Bennett linkage
    • Complex: M13 phage genome segment
    • Complex: Synthetic DNA oligonucleotides

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Supramolecule #1: DNA origami Bennett linkage

SupramoleculeName: DNA origami Bennett linkage / type: complex / ID: 1 / Parent: 0

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Supramolecule #2: M13 phage genome segment

SupramoleculeName: M13 phage genome segment / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Enterobacteria phage M13 (virus)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Synthetic DNA oligonucleotides

SupramoleculeName: Synthetic DNA oligonucleotides / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Details: Tris-Borate-EDTA (TBE) buffer containing 11 mM MgCl2
StainingType: POSITIVE / Material: Uranyl Formate
Details: The grid was washed twice by 1% (w/v) uranyl formate
GridModel: Homemade / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: LEICA EM GP
DetailsDNA origami Bennett linkage was diluted to ~2 nM with Tris-Borate-EDTA (TBE) buffer containing 11 mM MgCl2
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 0.69 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 19000
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsX-ray speckles in images were removed before CTF correction.
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 97.7 Å / Resolution method: FSC 0.5 CUT-OFF
Details: The 3D reconstruction was performed by using individual-particle electron tomography (IPET). The obtained 3D map was low-pass filtered to 8 nm
Number images used: 56
CTF correctionSoftware - Name: TOMOCTF
FSC plot (resolution estimation)

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