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- EMDB-0630: The axonemal doublet microtubule focusing on the inner junction r... -

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Entry
Database: EMDB / ID: EMD-0630
TitleThe axonemal doublet microtubule focusing on the inner junction region extracted from the cryo-electron tomography and subtomographic average of isolated Chlamydomonas pacrg; fap20 double mutant cilia
Map dataThe axonemal doublet microtubule focusing on the inner junction region extracted from the cryo-electron tomography and subtomographic average of isolated Chlamydomonas pacrg; fap20 double mutant cilia
Sample
  • Organelle or cellular component: The inner junction region of axonemal doublet microtubule averaged from Chlamydomonas pacrg; fap20 double mutant cilia
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 24.0 Å
AuthorsLin J / Fu G / Nicastro D / Dymek EE / Porter M / Smith EF
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR01GM112050 United States
National Institutes of Health/National Institute of General Medical SciencesR01GM055667 United States
National Institutes of Health/National Institute of General Medical SciencesR01GM083122 United States
CitationJournal: Mol Biol Cell / Year: 2019
Title: PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility.
Authors: Erin E Dymek / Jianfeng Lin / Gang Fu / Mary E Porter / Daniela Nicastro / Elizabeth F Smith /
Abstract: We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in ciliary assembly and motility, ...We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in ciliary assembly and motility, we used a combination of functional and structural studies, including newly identified mutants. Using cryo-electron tomography we show that PACRG and FAP20 form the inner junction between the A- and B-tubule along the length of all nine ciliary doublet microtubules. The lack of PACRG and FAP20 also results in reduced assembly of inner-arm dynein IDA and the beak-MIP structures. In addition, our functional studies reveal that loss of PACRG and/or FAP20 causes severe cell motility defects and reduced in vitro microtubule sliding velocities. Interestingly, the addition of exogenous PACRG and/or FAP20 protein to isolated mutant axonemes restores microtubule sliding velocities, but not ciliary beating. Taken together, these studies show that PACRG and FAP20 comprise the inner junction bridge that serves as a hub for both directly modulating dynein-driven microtubule sliding, as well as for the assembly of additional ciliary components that play essential roles in generating coordinated ciliary beating.
History
DepositionMar 5, 2019-
Header (metadata) releaseMar 27, 2019-
Map releaseJun 5, 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: 118
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 118
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0630.png

Downloads & links

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Map

FileDownload / File: emd_0630.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe axonemal doublet microtubule focusing on the inner junction region extracted from the cryo-electron tomography and subtomographic average of isolated Chlamydomonas pacrg; fap20 double mutant cilia
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.52 Å/pix.
x 95 pix.
= 524.685 Å		5.52 Å/pix.
x 70 pix.
= 386.61 Å		5.52 Å/pix.
x 80 pix.
= 441.84 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: 118 / Movie #1: 118
Minimum - Maximum109.69882 - 125.704895
Average (Standard dev.)116.70518 (±3.4906275)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-73-14-21
Dimensions708095
Spacing807095
CellA: 441.83997 Å / B: 386.61 Å / C: 524.685 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.5235.5235.523
M x/y/z807095
origin x/y/z0.0000.0000.000
length x/y/z441.840386.610524.685
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-14-73-21
NC/NR/NS807095
D min/max/mean109.699125.705116.705

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Supplemental data

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Sample components

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Entire : The inner junction region of axonemal doublet microtubule average...

EntireName: The inner junction region of axonemal doublet microtubule averaged from Chlamydomonas pacrg; fap20 double mutant cilia
Components
  • Organelle or cellular component: The inner junction region of axonemal doublet microtubule averaged from Chlamydomonas pacrg; fap20 double mutant cilia

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Supramolecule #1: The inner junction region of axonemal doublet microtubule average...

SupramoleculeName: The inner junction region of axonemal doublet microtubule averaged from Chlamydomonas pacrg; fap20 double mutant cilia
type: organelle_or_cellular_component / 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 for 1.5-2.5 seconds before plunging.
DetailsFreshly isolated and demembranated cilia from Chlamydomonas pacrg; fap20 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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMOD / Number subtomograms used: 1826
ExtractionNumber tomograms: 13 / Number images used: 1826 / Software - Name: MATLAB
Final angle assignmentType: OTHER / Software - Name: IMOD

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