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- EMDB-12475: A-brick 0.75:1 N:P PEG-oligolysine coated in PBS -

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Basic information

Entry
Database: EMDB / ID: EMD-12475
TitleA-brick 0.75:1 N:P PEG-oligolysine coated in PBS
Map dataRefined and post-processed map
Sample
  • Complex: A-brick 0.75:1 N:P PEG-oligolysine coated in PBS
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.29 Å
AuthorsBertosin E / Stoemmer P / Feigl E / Wenig M / Honemann MN / Dietz H
Funding support Germany, 4 items
OrganizationGrant numberCountry
German Research Foundation (DFG)Gottfried-Wilhelm-Leibniz Program Germany
European Research Council (ERC)Consolidator Grant (GA #724261) Germany
Max Planck SocietyMax Planck School Matter to Life Germany
German Research Foundation (DFG)SFB863 TPA9 Project ID 111166240 Germany
CitationJournal: ACS Nano / Year: 2021
Title: Cryo-Electron Microscopy and Mass Analysis of Oligolysine-Coated DNA Nanostructures.
Authors: Eva Bertosin / Pierre Stömmer / Elija Feigl / Maximilian Wenig / Maximilian N Honemann / Hendrik Dietz /
Abstract: Cationic coatings can enhance the stability of synthetic DNA objects in low ionic strength environments such as physiological fluids. Here, we used single-particle cryo-electron microscopy (cryo-EM), ...Cationic coatings can enhance the stability of synthetic DNA objects in low ionic strength environments such as physiological fluids. Here, we used single-particle cryo-electron microscopy (cryo-EM), pseudoatomic model fitting, and single-molecule mass photometry to study oligolysine and polyethylene glycol (PEG)-oligolysine-coated multilayer DNA origami objects. The coatings preserve coarse structural features well on a resolution of multiple nanometers but can also induce deformations such as twisting and bending. Higher-density coatings also led to internal structural deformations in the DNA origami test objects, in which a designed honeycomb-type helical lattice was deformed into a more square-lattice-like pattern. Under physiological ionic strength, where the uncoated objects disassembled, the coated objects remained intact but they shrunk in the helical direction and expanded in the direction perpendicular to the helical axis. Helical details like major/minor grooves and crossover locations were not discernible in cryo-EM maps that we determined of DNA origami coated with oligolysine and PEG-oligolysine, whereas these features were visible in cryo-EM maps determined from the uncoated reference objects. Blunt-ended double-helical interfaces remained accessible underneath the coating and may be used for the formation of multimeric DNA origami assemblies that rely on stacking interactions between blunt-ended helices. The ionic strength requirements for forming multimers from coated DNA origami differed from those needed for uncoated objects. Using single-molecule mass photometry, we found that the mass of coated DNA origami objects prior to and after incubation in low ionic strength physiological conditions remained unchanged. This finding indicated that the coating effectively prevented strand dissociation but also that the coating itself remained stable in place. Our results validate oligolysine coatings as a powerful stabilization method for DNA origami but also reveal several potential points of failure that experimenters should watch to avoid working with false premises.
History
DepositionFeb 24, 2021-
Header (metadata) releaseMar 31, 2021-
Map releaseMar 31, 2021-
UpdateJul 7, 2021-
Current statusJul 7, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0576
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0576
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12475.png

Downloads & links

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Map

FileDownload / File: emd_12475.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefined and post-processed map
Voxel sizeX=Y=Z: 4.638 Å
Density
Contour LevelBy AUTHOR: 0.0576 / Movie #1: 0.0576
Minimum - Maximum-0.06093953 - 0.26033232
Average (Standard dev.)0.0024458366 (±0.016384978)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 927.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.6384.6384.638
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z927.600927.600927.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0610.2600.002

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Supplemental data

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Additional map: Composite map from Multibody PostProcessing job

Fileemd_12475_additional_1.map
AnnotationComposite map from Multibody PostProcessing job
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : A-brick 0.75:1 N:P PEG-oligolysine coated in PBS

EntireName: A-brick 0.75:1 N:P PEG-oligolysine coated in PBS
Components
  • Complex: A-brick 0.75:1 N:P PEG-oligolysine coated in PBS

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Supramolecule #1: A-brick 0.75:1 N:P PEG-oligolysine coated in PBS

SupramoleculeName: A-brick 0.75:1 N:P PEG-oligolysine coated in PBS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#79
Source (natural)Organism: 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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 16.29 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 150955
FSC plot (resolution estimation)

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