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- EMDB-2949: Three Dimensional Dynamics and Fluctuations of DNA-Nanogold Dimer... -

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Entry
Database: EMDB / ID: EMD-2949
TitleThree Dimensional Dynamics and Fluctuations of DNA-Nanogold Dimers by Individual-Particle Electron Tomography
Map dataReconstruction of one particle of DNA-nanogold dimer by using individual-particle electron tomography.
Sample
  • Sample: Two 5-nm nanogolds bound to 84-base pair double-stranded DNA
  • DNA: double-stranded DNA
Keywords3D structure / DNA-nanogold conjugate / individual-particle electron tomography / IPET
Biological speciessynthetic construct (others)
Methodelectron tomography / negative staining / Resolution: 17.1 Å
AuthorsZhang L / Smith JM / Tong HM / Zhang X / Lei DS / Lu ZY / Alivisatos P / Ren G
CitationJournal: Nat Commun / Year: 2016
Title: Three-dimensional structural dynamics and fluctuations of DNA-nanogold conjugates by individual-particle electron tomography.
Authors: Lei Zhang / Dongsheng Lei / Jessica M Smith / Meng Zhang / Huimin Tong / Xing Zhang / Zhuoyang Lu / Jiankang Liu / A Paul Alivisatos / Gang Ren /
Abstract: DNA base pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA- ...DNA base pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of three-dimensional structure of DNA-nanocrystal-hybridized building blocks. Existing techniques do not allow for structural determination of these flexible and heterogeneous samples. Here we report cryo-electron microscopy and negative-staining electron tomography approaches to image, and three-dimensionally reconstruct a single DNA-nanogold conjugate, an 84-bp double-stranded DNA with two 5-nm nanogold particles for potential substrates in plasmon-coupling experiments. By individual-particle electron tomography reconstruction, we obtain 14 density maps at ∼2-nm resolution. Using these maps as constraints, we derive 14 conformations of dsDNA by molecular dynamics simulations. The conformational variation is consistent with that from liquid solution, suggesting that individual-particle electron tomography could be an expected approach to study DNA-assembling and flexible protein structure and dynamics.
History
DepositionMar 25, 2015-
Header (metadata) releaseMay 6, 2015-
Map releaseMar 30, 2016-
UpdateMar 30, 2016-
Current statusMar 30, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.299
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.299
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2949.tif

Downloads & links

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Map

FileDownload / File: emd_2949.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of one particle of DNA-nanogold dimer by using individual-particle electron tomography.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.88 Å/pix.
x 256 pix.
= 481.28 Å		1.88 Å/pix.
x 256 pix.
= 481.28 Å		1.88 Å/pix.
x 256 pix.
= 481.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.88 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.299 / Movie #1: 0.299
Minimum - Maximum-1.43568063 - 1.65837073
Average (Standard dev.)-0.00143957 (±0.08546878)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 481.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.881.881.88
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z481.280481.280481.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-1.4361.658-0.001

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Supplemental data

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Sample components

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Entire : Two 5-nm nanogolds bound to 84-base pair double-stranded DNA

EntireName: Two 5-nm nanogolds bound to 84-base pair double-stranded DNA
Components
  • Sample: Two 5-nm nanogolds bound to 84-base pair double-stranded DNA
  • DNA: double-stranded DNA

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Supramolecule #1000: Two 5-nm nanogolds bound to 84-base pair double-stranded DNA

SupramoleculeName: Two 5-nm nanogolds bound to 84-base pair double-stranded DNA
type: sample / ID: 1000
Details: 5 nm nanogold particles were stabilized via exchanging with bis-(p-sulfonatophenyl) phenylphosphine (BSPP). DNA sequences modified with a 5 thiol moiety were PAGE purified. DNA thiolated at ...Details: 5 nm nanogold particles were stabilized via exchanging with bis-(p-sulfonatophenyl) phenylphosphine (BSPP). DNA sequences modified with a 5 thiol moiety were PAGE purified. DNA thiolated at the 5 end was re-suspended in buffer (10mM Tris pH 8, 0.5mM EDTA). Nanogold particles and DNA were combined at a stoichiometric ratio of 1:2 in the presence of a reducing agent. Monoconjugates formed were separated by anion exchange HPLC, and the fractions concentrated by an Amicon Ultra spin filter, MW 100,000 (EMD Millipore Corp, Billerica, MA). Twenty microliters of nanogold monoconjugates, each containing complementary strands of DNA, were combined stoichiometrically as determined by absorption at 520 nm and allowed to react overnight at room temperature. The dimers were purified from unreacted monoconjugates by agarose gel electrophoresis.
Oligomeric state: Dimer / Number unique components: 2
Molecular weightExperimental: 52 KDa / Theoretical: 52 KDa / Method: Calculated from its sequence

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Macromolecule #1: double-stranded DNA

MacromoleculeName: double-stranded DNA / type: dna / ID: 1 / Name.synonym: dsDNA
Details: Two 5-nm nanogold bound to 84-base pair double-stranded DNA
Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
Molecular weightExperimental: 52 KDa / Theoretical: 52 KDa
SequenceString:
CCGGCGGCCC AGGTGTATCA GTGTTCGTTG CAAGCTCCAA CATCTGAGTA CCACGCATAC TATACTTGAA ATATCCGCGC CCGG

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 7.4
Details: 1X Dulbeccos phosphate-buffered saline (Invitrogen, La Jolla, CA), 2.7 mM KCl, 1.46 mM KH2PO4, 136.9 mM NaCl, and 8.1 mM Na2HPO4
StainingType: NEGATIVE
Details: EM Specimens were prepared by optimized negative-staining EM specimen preparation protocol as described (Zhang L. and Ren G, Journal of Lipid Research, (2010) 51, 1228-1236 and (2011) 52, ...Details: EM Specimens were prepared by optimized negative-staining EM specimen preparation protocol as described (Zhang L. and Ren G, Journal of Lipid Research, (2010) 51, 1228-1236 and (2011) 52, 175-84). In brief, nanogold-DNA dimer was diluted to 0.02 mg/ml with DPBS. Aliquots (about 4ul) were applied to the 200 mesh glow-discharged thin carbon-coated EM grids (Cu-200CN, Pacific Grid-Tech, USA). The grid was washed by deionized water for three times, and then washed by 1% uranyl formate for three times before blotting to drying.
GridDetails: 200 mesh glow-discharged thin carbon-coated EM grids (Cu-200CN, Pacific Grid-Tech, USA)
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeZEISS LIBRA120PLUS
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 125,000 times magnification
Specialist opticsEnergy filter - Name: ZEISS / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Detailstilt step is 1.5 degree
DateAug 16, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 81 / Average electron dose: 1000 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.2 µm / Nominal magnification: 125000
Sample stageSpecimen holder: Gatan / Specimen holder model: OTHER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 1.5 °

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Image processing

DetailsMicrographs were initially aligned together with the IMOD software package. The CTF was then corrected by TOMOCTF. The tilt series of particles in square windows of 512 pixels (~48 nm) were semi-automatically tracked and windowed by individual-particle electron tomography (IPET) software{eulerAnglesDetails}: Tomography tilt angle from -60 to 60 in step of 1.5
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.1 Å / Resolution method: OTHER / Software - Name: IPET, FETR, Spider, IMOD, EMAN, and, EMAN2
Details: Map was reconstructed by individual-particle electron tomography (IPET)and Focus ET Reconstruction Algorithm
Number images used: 81
CTF correctionDetails: TOMOCTF

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