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- EMDB-2023: Three-dimensional reconstruction of a targeted individual 17nm na... -

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Basic information

Entry
Database: EMDB / ID: EMD-2023
TitleThree-dimensional reconstruction of a targeted individual 17nm nascent high-density lipoprotein (HDL) particle by individual-particle electron tomography (IPET). The reconstruction displayed a ring-shaped structure of containing three protein - apoipoprotein A-Is (28 kDa each).
Map data17nm Nascent HDL particle Number 1 by cryo-ET
Sample
  • Sample: a 17nm nascent HDL particle
  • Protein or peptide: 17 nm nascent HDL
Keywordsreconstituted 17nm nascent high-density lipoprotein (HDl)
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / negative staining / Resolution: 42.0 Å
AuthorsZhang L / Ren G
CitationJournal: PLoS One / Year: 2012
Title: IPET and FETR: experimental approach for studying molecular structure dynamics by cryo-electron tomography of a single-molecule structure.
Authors: Lei Zhang / Gang Ren /
Abstract: The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional ...The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a "focused electron tomography reconstruction" (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single "snapshot" molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.
History
DepositionJan 4, 2012-
Header (metadata) releaseJan 20, 2012-
Map releaseJan 27, 2012-
UpdateMar 9, 2016-
Current statusMar 9, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2023.tif

Downloads & links

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Map

FileDownload / File: emd_2023.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation17nm Nascent HDL particle Number 1 by cryo-ET
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.46 Å/pix.
x 100 pix.
= 346. Å		3.46 Å/pix.
x 100 pix.
= 346. Å		3.46 Å/pix.
x 100 pix.
= 346. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.46 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.06166719 - 0.24149512
Average (Standard dev.)0.000000000028398 (±0.02020695)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 346.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.463.463.46
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z346.000346.000346.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-0.0620.2410.000

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Supplemental data

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Sample components

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Entire : a 17nm nascent HDL particle

EntireName: a 17nm nascent HDL particle
Components
  • Sample: a 17nm nascent HDL particle
  • Protein or peptide: 17 nm nascent HDL

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Supramolecule #1000: a 17nm nascent HDL particle

SupramoleculeName: a 17nm nascent HDL particle / type: sample / ID: 1000
Details: fresh sample was prepared with 2 days before using.
Oligomeric state: monomer / Number unique components: 1
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa

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Macromolecule #1: 17 nm nascent HDL

MacromoleculeName: 17 nm nascent HDL / type: protein_or_peptide / ID: 1 / Name.synonym: 17 nm nascent HDL
Details: HDL contains lipids and three apoA-I molecules. Each A-I molecular weight is about 28kDa.
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / Strain: nascent HDL / synonym: Human / Location in cell: Plasma
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsubtomogram averaging

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Sample preparation

Concentration0.01 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 standard cryo-EM protocol.
GridDetails: 200 mesh glow-discharged holey thin carbon-coated EM grids (Cu-200HN, Pacific Grid-Tech, USA)
VitrificationCryogen name: NITROGEN / Chamber humidity: 90 % / Chamber temperature: 103 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: manual / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI 12
TemperatureAverage: 100 K
DetailsTilt step is 1.5 degree
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 1.73 µm / Number real images: 81 / Average electron dose: 140 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: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 67000
Sample stageSpecimen holder: Gatan / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °

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Image processing

DetailsSingle targeted particle"s images were reconstructed by focus ET reconstruction (FETR) algorithm of individual particle electron tomography (IPET) method. Average number of tilts used in the 3D reconstructions: 81. Average tomographic tilt angle increment: 1.5.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 42.0 Å / Resolution method: OTHER / Software - Name: IPET, and, FETR
Details: Map was reconstructed by individual-particle electron tomography (IPET)and Focus ET Reconstruction Algorithm.
CTF correctionDetails: TOMOCTF
Final angle assignmentDetails: Tomography tilt angle from -60 to 60 in step of 1.5

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