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- EMDB-3787: Cryo-electron tomography averaged map of microtubule doublet from... -

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Basic information

Entry
Database: EMDB / ID: 3787
TitleCryo-electron tomography averaged map of microtubule doublet from p23 mutant Chlamydomonas axoneme
Map dataSubaverage after subtomogram classification of Chlamydomonas pf23 cilia, based on outer arm dynein structure.
SampleCiliary microtubule doublet with dynein, radial spokes and other binding proteins:
SourceChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / 50 Å resolution
AuthorsSale WS / Ishikawa T / Yamamoto R / Obbineni JM / Alford LM / Hwang J / Ide T / Owa M / Kon T / Inaba K / Noliyanda J / King SM / Dutcher S
CitationJournal: PLoS Genet. / Year: 2017
Title: Chlamydomonas DYX1C1/PF23 is essential for axonemal assembly and proper morphology of inner dynein arms.
Authors: Ryosuke Yamamoto / Jagan M Obbineni / Lea M Alford / Takahiro Ide / Mikito Owa / Juyeon Hwang / Takahide Kon / Kazuo Inaba / Noliyanda James / Stephen M King / Takashi Ishikawa / Winfield S Sale / Susan K Dutcher
Abstract: Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we ...Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner dynein arms (IDA) as well as a fraction of the outer dynein arms (ODA). A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary dyneins are reduced compared to wild-type, which suggests an important role in dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly.
DateDeposition: Jul 6, 2017 / Header (metadata) release: Aug 16, 2017 / Map release: Sep 6, 2017 / Last update: Sep 20, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: -0.23
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: -0.23
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3787.map.gz (map file in CCP4 format, 49949 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
232 pix
5.68 Å/pix.
= 1317.76 Å
232 pix
5.68 Å/pix.
= 1317.76 Å
232 pix
5.68 Å/pix.
= 1317.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.68 Å
Density
Contour Level:0.001 (by author), -0.23 (movie #1):
Minimum - Maximum-0.70259494 - 0.6101861
Average (Standard dev.)0.000158723 (0.091841586)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions232232232
Origin000
Limit231231231
Spacing232232232
CellA=B=C: 1317.76 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.685.685.68
M x/y/z232232232
origin x/y/z0.0000.0000.000
length x/y/z1317.7601317.7601317.760
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS232232232
D min/max/mean-0.7030.6100.000

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Supplemental data

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Sample components

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Entire Ciliary microtubule doublet with dynein, radial spokes and other ...

EntireName: Ciliary microtubule doublet with dynein, radial spokes and other binding proteins
Number of components: 1

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Component #1: cellular-component, Ciliary microtubule doublet with dynein, radi...

Cellular-componentName: Ciliary microtubule doublet with dynein, radial spokes and other binding proteins
Recombinant expression: No
SourceSpecies: Chlamydomonas reinhardtii (plant) / Strain: pf23
Source (natural)Organelle: cilia

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Experimental details

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Sample preparation

SpecimenSpecimen state: cell / Method: cryo EM
Sample solutionBuffer solution: 30 mM HEPES, 5 mM MgSO4, 1 mM DTT, 1 mM EGTA, and 50 mM potassium acetate, pH7.4
pH: 7.4
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 70 % / Details: one side blotting

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Electron microscopy imaging

ImagingMicroscope: JEOL 2200FS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 0.8 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 20000 X (nominal) / Imaging mode: BRIGHT FIELD / Energy filter: JEOL in-column / Energy window: 0-25 eV
Specimen HolderModel: GATAN LIQUID NITROGEN / Tilt Angle: -60 - 60 deg.
CameraDetector: TVIPS TEMCAM-F416 (4k x 4k)

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Image processing

ProcessingMethod: subtomogram averaging
3D reconstructionAlgorithm: BACK PROJECTION / Software: SPIDER / Resolution: 50 Å / Resolution method: OTHER
Details: Visual estimation, compared with datasets in the past
Euler angles: 3D cross correlation by Spider

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