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- EMDB-20166: The central pair apparatus focusing on the C1a-e-c supercomplex e... -

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Basic information

Entry
Database: EMDB / ID: EMD-20166
TitleThe central pair apparatus focusing on the C1a-e-c supercomplex extracted from the cryo-electron tomography and subtomographic average of isolated Chlamydomonas fap76-1,fap81 double mutant axoneme
Map dataThe central pair apparatus focusing on the C1a-e-c supercomplex extracted from the cryo-electron tomography and subtomographic average of isolated Chlamydomonas fap76-1,fap81 double mutant axoneme
Sample
  • Complex: The C1a-e-c supercomplex of central pair apparatus averaged from Chlamydomonas fap76-1,fap81 double mutant cilia
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 23.0 Å
AuthorsFu G / Nicastro D
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR01GM083122 United States
National Institutes of Health/National Institute of General Medical SciencesR37GM030626 United States
National Institutes of Health/National Institute of General Medical SciencesR01GM112050 United States
National Institutes of Health/National Institute of General Medical SciencesR35GM122574 United States
CitationJournal: J Cell Biol / Year: 2019
Title: Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus.
Authors: Gang Fu / Lei Zhao / Erin Dymek / Yuqing Hou / Kangkang Song / Nhan Phan / Zhiguo Shang / Elizabeth F Smith / George B Witman / Daniela Nicastro /
Abstract: Nearly all motile cilia contain a central apparatus (CA) composed of two connected singlet microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA ...Nearly all motile cilia contain a central apparatus (CA) composed of two connected singlet microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild-type with CA mutants. We identified a large (>2 MD) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and 3D structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating.
History
DepositionApr 26, 2019-
Header (metadata) releaseMay 22, 2019-
Map releaseNov 13, 2019-
UpdateAug 12, 2020-
Current statusAug 12, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 124
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 124
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20166.png

Downloads & links

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Map

FileDownload / File: emd_20166.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe central pair apparatus focusing on the C1a-e-c supercomplex extracted from the cryo-electron tomography and subtomographic average of isolated Chlamydomonas fap76-1,fap81 double mutant axoneme
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.52 Å/pix.
x 120 pix.
= 662.76 Å		5.52 Å/pix.
x 90 pix.
= 497.07 Å		5.52 Å/pix.
x 90 pix.
= 497.07 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: 5.523 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 124 / Movie #1: 124
Minimum - Maximum114.09506 - 133.99298
Average (Standard dev.)122.53439 (±3.0128028)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-19-102-19
Dimensions9090120
Spacing9090120
CellA: 497.06998 Å / B: 497.06998 Å / C: 662.75995 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.5235.5235.523
M x/y/z9090120
origin x/y/z0.0000.0000.000
length x/y/z497.070497.070662.760
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ254265109
MAP C/R/S123
start NC/NR/NS-102-19-19
NC/NR/NS9090120
D min/max/mean114.095133.993122.534

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Supplemental data

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Sample components

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Entire : The C1a-e-c supercomplex of central pair apparatus averaged from ...

EntireName: The C1a-e-c supercomplex of central pair apparatus averaged from Chlamydomonas fap76-1,fap81 double mutant cilia
Components
  • Complex: The C1a-e-c supercomplex of central pair apparatus averaged from Chlamydomonas fap76-1,fap81 double mutant cilia

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Supramolecule #1: The C1a-e-c supercomplex of central pair apparatus averaged from ...

SupramoleculeName: The C1a-e-c supercomplex of central pair apparatus averaged from Chlamydomonas fap76-1,fap81 double mutant cilia
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Organelle: cilia

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER
Details: back-side blotting with No.1 Whatman filter for 1.5-2.5 seconds before plunging.
DetailsFreshly isolated and demembranated cilia from Chlamydomonas fap76-1,fap81 double mutant cells

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-15 / Average exposure time: 6.0 sec. / Average electron dose: 1.57 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 0.5 µm / Calibrated defocus min: 0.2 µm / Calibrated magnification: 26000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMOD / Number subtomograms used: 1018
ExtractionNumber tomograms: 21 / Number images used: 1018 / Software - Name: MATLAB
Final angle assignmentType: OTHER

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