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- EMDB-7231: Single-Molecule 3D Image of DNA Origami Bennett Linkage by Indivi... -

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Basic information

Entry
Database: EMDB / ID: EMD-7231
TitleSingle-Molecule 3D Image of DNA Origami Bennett Linkage by Individual Particle Electron Tomography (No. 023)
Map dataprimary map
Sample
  • Complex: DNA origami Bennett linkage
    • Complex: M13 phage genome segment
    • Complex: Synthetic DNA oligonucleotides
Biological speciesEscherichia virus M13 / synthetic construct (others)
Methodelectron tomography / cryo EM / negative staining / Resolution: 62.6 Å
AuthorsLei D / Marras A / Liu J / Huang C / Zhou L / Castro C / Su H / Ren G
Funding support United States, 4 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DMR-1344290 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM104427 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL115153 United States
Department of Energy (DOE, United States)DE-AC02-05CH11231 United States
CitationJournal: Nat Commun / Year: 2018
Title: Three-dimensional structural dynamics of DNA origami Bennett linkages using individual-particle electron tomography.
Authors: Dongsheng Lei / Alexander E Marras / Jianfang Liu / Chao-Min Huang / Lifeng Zhou / Carlos E Castro / Hai-Jun Su / Gang Ren /
Abstract: Scaffolded DNA origami has proven to be a powerful and efficient technique to fabricate functional nanomachines by programming the folding of a single-stranded DNA template strand into three- ...Scaffolded DNA origami has proven to be a powerful and efficient technique to fabricate functional nanomachines by programming the folding of a single-stranded DNA template strand into three-dimensional (3D) nanostructures, designed to be precisely motion-controlled. Although two-dimensional (2D) imaging of DNA nanomachines using transmission electron microscopy and atomic force microscopy suggested these nanomachines are dynamic in 3D, geometric analysis based on 2D imaging was insufficient to uncover the exact motion in 3D. Here we use the individual-particle electron tomography method and reconstruct 129 density maps from 129 individual DNA origami Bennett linkage mechanisms at ~ 6-14 nm resolution. The statistical analyses of these conformations lead to understanding the 3D structural dynamics of Bennett linkage mechanisms. Moreover, our effort provides experimental verification of a theoretical kinematics model of DNA origami, which can be used as feedback to improve the design and control of motion via optimized DNA sequences and routing.
History
DepositionDec 6, 2017-
Header (metadata) releaseJan 24, 2018-
Map releaseFeb 21, 2018-
UpdateDec 25, 2019-
Current statusDec 25, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.326
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.326
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7231.png

Downloads & links

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Map

FileDownload / File: emd_7231.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationprimary map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.8 Å/pix.
x 192 pix.
= 921.6 Å		4.8 Å/pix.
x 192 pix.
= 921.6 Å		4.8 Å/pix.
x 192 pix.
= 921.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelMovie #1: 0.326
Minimum - Maximum-0.2179426 - 1.4913394
Average (Standard dev.)0.014540869 (±0.10220008)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 921.60004 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.84.84.8
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z921.600921.600921.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ401401401
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-0.2181.4910.015

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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: Escherichia virus M13
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: NEGATIVE / Material: Uranyl Formate
Details: The grid was washed twice by 1% (w/v) uranyl formate
GridMaterial: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / 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

MicroscopeZEISS LIBRA120PLUS
Specialist opticsEnergy filter - Name: In-column Omega Filter
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 1.17 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN CT3500 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

DetailsX-ray speckles in images were removed before alignment and 3D reconstruction.
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 62.6 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 Gaussian low-pass filtered to 8 nm
Number images used: 59
FSC plot (resolution estimation)

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