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- EMDB-9262: Single-Molecule 3D Image of Double Complexes of DNA-Nanogold Conj... -

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Basic information

Entry
Database: EMDB / ID: EMD-9262
TitleSingle-Molecule 3D Image of Double Complexes of DNA-Nanogold Conjugates (No. 01)
Map dataDouble Complexes of DNA-Nanogold Conjugates (No. 01)
Sample
  • Complex: Double complexes of 84-base pair double-stranded DNA bound with two 5-nm nanogolds
Biological speciessynthetic construct (others)
Methodelectron tomography / negative staining / Resolution: 17.6 Å
AuthorsWu H / Zhai X / Lei D / Liu J / Yu Y / Bie R / Ren G
CitationJournal: Sci Rep / Year: 2018
Title: An Algorithm for Enhancing the Image Contrast of Electron Tomography.
Authors: Hao Wu / Xiaobo Zhai / Dongsheng Lei / Jianfang Liu / Yadong Yu / Rongfang Bie / Gang Ren /
Abstract: Three-dimensional (3D) reconstruction of a single protein molecule is essential for understanding the relationship between the structural dynamics and functions of the protein. Electron tomography ...Three-dimensional (3D) reconstruction of a single protein molecule is essential for understanding the relationship between the structural dynamics and functions of the protein. Electron tomography (ET) provides a tool for imaging an individual particle of protein from a series of tilted angles. Individual-particle electron tomography (IPET) provides an approach for reconstructing a 3D density map from a single targeted protein particle (without averaging from different particles of this type of protein), in which the target particle was imaged from a series of tilting angles. However, owing to radiation damage limitations, low-dose images (high noise, and low image contrast) are often challenging to be aligned for 3D reconstruction at intermediate resolution (1-3 nm). Here, we propose a computational method to enhance the image contrast, without increasing any experimental dose, for IPET 3D reconstruction. Using an edge-preserving smoothing-based multi-scale image decomposition algorithm, this method can detect the object against a high-noise background and enhance the object image contrast without increasing the noise level or significantly decreasing the image resolution. The method was validated by using both negative staining (NS) ET and cryo-ET images. The successful 3D reconstruction of a small molecule (<100 kDa) indicated that this method can be used as a supporting tool to current ET 3D reconstruction methods for studying protein dynamics via structure determination from each individual particle of the same type of protein.
History
DepositionOct 26, 2018-
Header (metadata) releaseNov 28, 2018-
Map releaseDec 12, 2018-
UpdateDec 12, 2018-
Current statusDec 12, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.15
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.15
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9262.png

Downloads & links

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Map

FileDownload / File: emd_9262.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDouble Complexes of DNA-Nanogold Conjugates (No. 01)
Voxel sizeX=Y=Z: 2.82 Å
Density
Contour LevelMovie #1: 0.15
Minimum - Maximum-0.820532 - 1.4094514
Average (Standard dev.)-0.00022082565 (±0.028251465)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-150-150-150
Dimensions300300300
Spacing300300300
CellA=B=C: 846.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.822.822.82
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z846.000846.000846.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS-150-150-150
NC/NR/NS300300300
D min/max/mean-0.8211.409-0.000

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Supplemental data

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Sample components

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Entire : Double complexes of 84-base pair double-stranded DNA bound with t...

EntireName: Double complexes of 84-base pair double-stranded DNA bound with two 5-nm nanogolds
Components
  • Complex: Double complexes of 84-base pair double-stranded DNA bound with two 5-nm nanogolds

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Supramolecule #1: Double complexes of 84-base pair double-stranded DNA bound with t...

SupramoleculeName: Double complexes of 84-base pair double-stranded DNA bound with two 5-nm nanogolds
type: complex / ID: 1 / Parent: 0
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.
Source (natural)Organism: synthetic construct (others)

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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
Details: 1X Dulbeccos phosphate-buffered saline, 2.7 mM KCl, 1.46 mM KH2PO4, 136.9 mM NaCl, and 8.1 mM Na2HPO4
StainingType: NEGATIVE / Material: Uranyl Formate
Details: The grid was stained with 1% (w/v) uranyl formate by using optimized negative-staining (OpNS) protocol.
GridModel: Homemade / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
Details5-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.
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeZEISS LIBRA120PLUS
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal magnification: 125000
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 54.64 e/Å2

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Image processing

CTF correctionSoftware - Name: TOMOCTF
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 17.6 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 low-pass filtered to 1.6 nm.
Number images used: 67
DetailsX-ray speckles in images were removed before CTF correction.
FSC plot (resolution estimation)

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