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- EMDB-0629: The axonemal doublet microtubule focusing on the inner junction r... -

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Entry
Database: EMDB / ID: EMD-0629
TitleThe axonemal doublet microtubule focusing on the inner junction region extracted from the cryo-electron tomography and subtomographic average of isolated Chlamydomonas pacrg mutant cilia
Map data
SampleThe inner junction region of axonemal doublet microtubule averaged from Chlamydomonas pacrg mutant cilia
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 27 Å
AuthorsLin J / Fu G / Nicastro D / Dymek EE / Porter M / Smith EF
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 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, as well as reduced 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. Video S1 Video S1 . Video of a wild-type cell swimming forward with an asymmetric waveform. Cells were captured at 500 fps using a pco.1200HS camera and videos created at 30 fps with Camware (The Cooke Corporation, Londonderry, NH) and ImageJ (Schneider , 2012) software. Video S2 Video S2 . Video of a mutant cell (CLiP 159) with abnormal waveform and motility. Cells were captured at 500 fps using a pco.1200HS camera and videos created at 30 fps with Camware (The Cooke Corporation, Londonderry, NH) and ImageJ (Schneider , 2012) software. Video S3 Video S3 . Video of a mutant cell (-cc1031) with abnormal waveform and motility. Cells were captured at 500 fps using a pco.1200HS camera and videos created at 30 fps with Camware (The Cooke Corporation, Londonderry, NH) and ImageJ (Schneider , 2012) software.
DateDeposition: Mar 4, 2019 / Header (metadata) release: Mar 27, 2019 / Map release: Jun 5, 2019 / Update: Jun 5, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 131
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 131
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0629.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
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
Contour LevelBy AUTHOR: 131.0 / Movie #1: 131
Minimum - Maximum125.946395999999993 - 137.419279999999986
Average (Standard dev.)131.141310000000004 (±2.0744257)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-70-14-21
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-14-70-21
NC/NR/NS807095
D min/max/mean125.946137.419131.141

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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 mutant cilia
Number of components: 1

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Component #1: cellular-component, The inner junction region of axonemal doublet...

Cellular-componentName: The inner junction region of axonemal doublet microtubule averaged from Chlamydomonas pacrg mutant cilia
Recombinant expression: No
SourceSpecies: Chlamydomonas reinhardtii (plant)
Source (natural)Organelle: cilia

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Experimental details

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Sample preparation

SpecimenSpecimen state: Cell / Method: cryo EM
Sample solutionpH: 7.4
Support filmunspecified
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 298 K
Details: back-side blotting for 1.5-2.5 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.57 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 26000.0 X (calibrated) / Imaging mode: BRIGHT FIELD / Energy filter: GIF Bioquantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: subtomogram averaging / Applied symmetry: C1 (asymmetric) / Number of subtomograms: 2333
3D reconstructionSoftware: IMOD / Resolution: 27 Å / Resolution method: FSC 0.5 CUT-OFF

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