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- EMDB-2960: Three Dimensional Dynamics and Fluctuations of DNA-Nanogold Dimer... -
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Basic information
Entry | Database: EMDB / ID: EMD-2960 | |||||||||
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Title | Three Dimensional Dynamics and Fluctuations of DNA-Nanogold Dimers by Individual-Particle Electron Tomography | |||||||||
![]() | Reconstruction of one particle of DNA-nanogold dimer by using individual-particle electron tomography. | |||||||||
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![]() | 3D structure / DNA-nanogold conjugate / individual-particle electron tomography / IPET | |||||||||
Biological species | synthetic construct (others) | |||||||||
Method | electron tomography / negative staining / Resolution: 17.9 Å | |||||||||
![]() | Zhang L / Smith JM / Tong HM / Zhang X / Lei DS / Lu ZY / Alivisatos P / Ren G | |||||||||
![]() | ![]() 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. | |||||||||
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Structure visualization
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Map data | ![]() | 56.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 12.3 KB 12.3 KB | Display Display | ![]() |
Images | ![]() | 192.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 2948C ![]() 2949C ![]() 2950C ![]() 2951C ![]() 2952C ![]() 2953C ![]() 2954C ![]() 2955C ![]() 2956C ![]() 2957C ![]() 2958C ![]() 2959C ![]() 2961C C: citing same article ( |
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Similar structure data |
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Links
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Related items in Molecule of the Month |
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Map
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Annotation | Reconstruction of one particle of DNA-nanogold dimer by using individual-particle electron tomography. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.88 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Two 5-nm nanogolds bound to 84-base pair double-stranded DNA
Entire | Name: Two 5-nm nanogolds bound to 84-base pair double-stranded DNA |
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Components |
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-Supramolecule #1000: Two 5-nm nanogolds bound to 84-base pair double-stranded DNA
Supramolecule | Name: 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 |
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Molecular weight | Experimental: 52 KDa / Theoretical: 52 KDa / Method: Calculated from its sequence |
-Macromolecule #1: double-stranded DNA
Macromolecule | Name: 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 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Experimental: 52 KDa / Theoretical: 52 KDa |
Sequence | String: CCGGCGGCCC AGGTGTATCA GTGTTCGTTG CAAGCTCCAA CATCTGAGTA CCACGCATAC TATACTTGAA ATATCCGCGC CCGG |
-Experimental details
-Structure determination
Method | negative staining |
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![]() | electron tomography |
Aggregation state | particle |
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Sample preparation
Concentration | 0.02 mg/mL |
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Buffer | pH: 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 |
Staining | Type: 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. |
Grid | Details: 200 mesh glow-discharged thin carbon-coated EM grids (Cu-200CN, Pacific Grid-Tech, USA) |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
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Electron microscopy
Microscope | ZEISS LIBRA120PLUS |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 125,000 times magnification |
Specialist optics | Energy filter - Name: ZEISS / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV |
Details | tilt step is 1.5 degree |
Date | Aug 16, 2012 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 89 / Average electron dose: 1000 e/Å2 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Illumination 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 stage | Specimen holder: Gatan / Specimen holder model: OTHER / Tilt series - Axis1 - Min angle: -66 ° / Tilt series - Axis1 - Max angle: 66 ° / Tilt series - Axis1 - Angle increment: 1.5 ° |
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Image processing
Details | Micrographs 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 -66 to 66 in step of 1.5 |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.9 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: 89 |
CTF correction | Details: TOMOCTF |