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- EMDB-6159: Detailed Structural and biochemical characterization of the nexin... -

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Basic information

Entry
Database: EMDB / ID: EMD-6159
TitleDetailed Structural and biochemical characterization of the nexin-dynein regulatory complex
Map dataStreptavidin-labeled N-DRC structure from DRC2-M-BCCP axoneme
Sample
  • Sample: Streptavidin-labeled N-DRC structure from DRC2-M-BCCP axoneme
  • Organelle or cellular component: axoneme
Keywordscilia and flagella / axoneme / dynein regulatory complex
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 45.0 Å
AuthorsOda T / Yanagisawa H / Kikkawa M
CitationJournal: Mol Biol Cell / Year: 2015
Title: Detailed structural and biochemical characterization of the nexin-dynein regulatory complex.
Authors: Toshiyuki Oda / Haruaki Yanagisawa / Masahide Kikkawa /
Abstract: The nexin-dynein regulatory complex (N-DRC) forms a cross-bridge between the outer doublet microtubules of the axoneme and regulates dynein motor activity in cilia/flagella. Although the molecular ...The nexin-dynein regulatory complex (N-DRC) forms a cross-bridge between the outer doublet microtubules of the axoneme and regulates dynein motor activity in cilia/flagella. Although the molecular composition and the three-dimensional structure of N-DRC have been studied using mutant strains lacking N-DRC subunits, more accurate approaches are necessary to characterize the structure and function of N-DRC. In this study, we precisely localized DRC1, DRC2, and DRC4 using cryo-electron tomography and structural labeling. All three N-DRC subunits had elongated conformations and spanned the length of N-DRC. Furthermore, we purified N-DRC and characterized its microtubule-binding properties. Purified N-DRC bound to the microtubule and partially inhibited microtubule sliding driven by the outer dynein arms (ODAs). Of interest, microtubule sliding was observed even in the presence of fourfold molar excess of N-DRC relative to ODA. These results provide insights into the role of N-DRC in generating the beating motions of cilia/flagella.
History
DepositionOct 25, 2014-
Header (metadata) releaseDec 3, 2014-
Map releaseDec 3, 2014-
UpdateMay 25, 2016-
Current statusMay 25, 2016Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 30.7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 30.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6159.gif

Downloads & links

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Map

FileDownload / File: emd_6159.map.gz / Format: CCP4 / Size: 3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStreptavidin-labeled N-DRC structure from DRC2-M-BCCP axoneme
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.07 Å/pix.
x 90 pix.
= 546.3 Å		6.07 Å/pix.
x 80 pix.
= 485.6 Å		6.07 Å/pix.
x 110 pix.
= 667.7 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 6.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 30.699999999999999 / Movie #1: 30.7
Minimum - Maximum-180.905090330000007 - 200.319183350000003
Average (Standard dev.)0.00166648 (±69.002189639999997)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions8011090
Spacing8011090
CellA: 667.7 Å / B: 485.6 Å / C: 546.3 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.076.076.07
M x/y/z1108090
origin x/y/z0.0000.0000.000
length x/y/z667.700485.600546.300
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS1108090
D min/max/mean-180.905200.3190.002

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Supplemental data

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Sample components

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Entire : Streptavidin-labeled N-DRC structure from DRC2-M-BCCP axoneme

EntireName: Streptavidin-labeled N-DRC structure from DRC2-M-BCCP axoneme
Components
  • Sample: Streptavidin-labeled N-DRC structure from DRC2-M-BCCP axoneme
  • Organelle or cellular component: axoneme

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Supramolecule #1000: Streptavidin-labeled N-DRC structure from DRC2-M-BCCP axoneme

SupramoleculeName: Streptavidin-labeled N-DRC structure from DRC2-M-BCCP axoneme
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) / Organelle: flagella

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.2
Details: 30 mM Hepes-NaOH pH 7.2, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, 50 mM NaCl
GridDetails: 300 mesh copper grid, holey carbon
VitrificationCryogen name: ETHANE / Chamber humidity: 98 % / Chamber temperature: 93 K / Instrument: LEICA EM GP / Method: Blot for 5 seconds before plunging

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Electron microscopy

MicroscopeJEOL 3100FFC
Specialist opticsEnergy filter - Name: Omega / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateMar 19, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 100 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 7.0 µm / Nominal defocus min: 6.0 µm / Nominal magnification: 25700
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -65 ° / Tilt series - Axis1 - Max angle: 65 °

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Image processing

DetailsNumber of tilts (projections) used in 3D reconstruction: 65 Tomographic tilt angle increment: 2
Final reconstructionResolution.type: BY AUTHOR / Resolution: 45.0 Å / Resolution method: OTHER / Software - Name: IMOD, PEET

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