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- EMDB-5712: Novel Structural Labeling Method using Cryo-electron Tomography a... -

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Basic information

Entry
Database: EMDB / ID: EMD-5712
TitleNovel Structural Labeling Method using Cryo-electron Tomography and Biotin-Streptavidin System
Map dataAveraged subtomogram of Chlamydomonas axoneme
Sample
  • Sample: Chlamydomonas axoneme, wild type
  • Organelle or cellular component: Axoneme
KeywordsCryo-electron tomography / Chlamydomonas reinhardtii / biotin-streptavidin / cilia and flagella / dynein / structural labeling
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 60.0 Å
AuthorsOda T / Kikkawa M
CitationJournal: J Struct Biol / Year: 2013
Title: Novel structural labeling method using cryo-electron tomography and biotin-streptavidin system.
Authors: Toshiyuki Oda / Masahide Kikkawa /
Abstract: There are a number of large macromolecular complexes that play important roles in the cell, and identifying the positions of their components is a key step to understanding their structure and ...There are a number of large macromolecular complexes that play important roles in the cell, and identifying the positions of their components is a key step to understanding their structure and function. Several structural labeling methods have been applied to electron microscopy in order to locate a specific component within a macromolecular complex, but each method is associated with problems in specificity, occupancy, signal intensity or precision. Here, we report a novel method for identifying the 3D locations of proteins using biotin-streptavidin labeling and cryo-electron tomography. We labeled a biotinylation-tagged intermediate chain of an axonemal dynein by streptavidin within the Chlamydomonas axoneme and visualized the 3D positions of the labels using subtomogram averaging. Increase of the density attributed to the bound streptavidin was validated by Student's t-test. In conclusion, the combination of the biotin-streptavidin system and cryo-electron tomography is a powerful method to investigate the structure of large macromolecular complexes.
History
DepositionJul 1, 2013-
Header (metadata) releaseJul 24, 2013-
Map releaseJul 24, 2013-
UpdateApr 23, 2014-
Current statusApr 23, 2014Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 34419
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 34419
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5712_1.tif

Downloads & links

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Map

FileDownload / File: emd_5712.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAveraged subtomogram of Chlamydomonas axoneme
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.07 Å/pix.
x 240 pix.
= 1456.8 Å		6.07 Å/pix.
x 240 pix.
= 1456.8 Å		6.07 Å/pix.
x 240 pix.
= 1456.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 6.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 34419.0 / Movie #1: 34419
Minimum - Maximum0.0023676 - 40496.015625
Average (Standard dev.)11625.948242189999291 (±14810.637695310000709)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 1456.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.076.076.07
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z1456.8001456.8001456.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean0.00240496.01611625.948

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Supplemental data

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Sample components

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Entire : Chlamydomonas axoneme, wild type

EntireName: Chlamydomonas axoneme, wild type
Components
  • Sample: Chlamydomonas axoneme, wild type
  • Organelle or cellular component: Axoneme

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Supramolecule #1000: Chlamydomonas axoneme, wild type

SupramoleculeName: Chlamydomonas axoneme, wild type / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Axoneme

SupramoleculeName: Axoneme / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: 137c / Organelle: flagella

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

Concentration0.04 mg/mL
BufferpH: 7.2
Details: 30 mM Hepes-NaOH pH 7.2, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, 50 mM CH3COOK, and 1 mM phenylmethylsulfonyl fluoride
GridDetails: 300 mesh copper grid with home-made holey carbon support
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 93 K / Instrument: LEICA EM GP / Method: Blot for 5 seconds before plunging

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Electron microscopy

MicroscopeJEOL 3100FFC
TemperatureMin: 90 K / Max: 95 K
DateJan 16, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 90 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 25700 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal magnification: 15000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60.5 ° / Tilt series - Axis1 - Max angle: 60.5 °

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 60.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PEET, Ruby-Helix / Number subtomograms used: 77

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