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- EMDB-0029: Sequence-programmable covalent bonding of designed DNA assemblies... -

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Basic information

Entry
Database: EMDB / ID: EMD-0029
TitleSequence-programmable covalent bonding of designed DNA assemblies, brick-like_object_with_TT-motifs_(1)-(4)_crosslinked
Map data
Sample
  • Complex: DNA-Origami
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 22.0 Å
AuthorsDietz H / Kube M
CitationJournal: Sci Adv / Year: 2018
Title: Sequence-programmable covalent bonding of designed DNA assemblies.
Authors: Thomas Gerling / Massimo Kube / Benjamin Kick / Hendrik Dietz /
Abstract: Bottom-up fabrication of custom nanostructures using the methods of DNA nanotechnology has great potential for applications in many areas of science and technology. One obstacle to applications ...Bottom-up fabrication of custom nanostructures using the methods of DNA nanotechnology has great potential for applications in many areas of science and technology. One obstacle to applications concerns the constrained environmental conditions at which DNA objects retain their structure. We present a general, site-selective, and scalable method for creating additional covalent bonds that increase the structural stability of DNA nanostructures. Placement of thymidines in close proximity within DNA nanostructures allows the rational creation of sites for covalent cyclobutane pyrimidine dimer (CPD) bonds induced via ultraviolet irradiation. The additional covalent bonds may be used in a sequence-programmable fashion to link free strand termini, to bridge strand breaks at crossover sites, and to create additional interhelical connections. Thus designed multilayer DNA origami objects can remain stable at temperatures up to 90°C and in pure double-distilled water with no additional cations present. In addition, these objects show enhanced resistance against nuclease activity. Cryo-electron microscopy (cryo-EM) structural analysis of non-cross-linked and cross-linked objects indicated that the global shape and the internal network of crossovers are preserved after irradiation. A cryo-EM map of a CPD-stabilized multilayer DNA origami object determined at physiological ionic strength reveals a substantial swelling behavior, presumably caused by repulsive electrostatic forces that, without covalent stabilization, would cause disassembly at low ionic strength. Our method opens new avenues for applications of DNA nanostructures in a wider range of conditions.
History
DepositionMay 23, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseAug 29, 2018-
UpdateAug 29, 2018-
Current statusAug 29, 2018Processing site: PDBe / Status: Released

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Structure visualization

Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0029.png

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Map

FileDownload / File: emd_0029.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.3 Å/pix.
x 400 pix.
= 920. Å		2.3 Å/pix.
x 400 pix.
= 920. Å		2.3 Å/pix.
x 400 pix.
= 920. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.059
Minimum - Maximum-0.2717565 - 0.29781535
Average (Standard dev.)0.0009789545 (±0.016070727)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 920.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : DNA-Origami

EntireName: DNA-Origami
Components
  • Complex: DNA-Origami

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Supramolecule #1: DNA-Origami

SupramoleculeName: DNA-Origami / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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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

BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 33000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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