|Entry||Database: EMDB / ID: EMD-0629|
|Title||The axonemal doublet microtubule focusing on the inner junction region extracted from the cryo-electron tomography and subtomographic average of isolated Chlamydomonas pacrg mutant cilia|
|Sample||The inner junction region of axonemal doublet microtubule averaged from Chlamydomonas pacrg mutant cilia|
|Biological species||Chlamydomonas reinhardtii (plant)|
|Method||subtomogram averaging / cryo EM / Resolution: 27 Å|
|Authors||Lin J / Fu G / Nicastro D / Dymek EE / Porter M / Smith EF|
|Citation||Journal: 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.
|Date||Deposition: Mar 4, 2019 / Header (metadata) release: Mar 27, 2019 / Map release: Jun 5, 2019 / Update: Jun 5, 2019|
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_0629.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
generated in cubic-lattice coordinate
|Voxel size||X=Y=Z: 5.523 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire The inner junction region of axonemal doublet microtubule average...
|Entire||Name: The inner junction region of axonemal doublet microtubule averaged from Chlamydomonas pacrg mutant cilia|
Number of components: 1
-Component #1: cellular-component, The inner junction region of axonemal doublet...
|Cellular-component||Name: The inner junction region of axonemal doublet microtubule averaged from Chlamydomonas pacrg mutant cilia|
Recombinant expression: No
|Source||Species: Chlamydomonas reinhardtii (plant)|
|Source (natural)||Organelle: cilia|
|Specimen||Specimen state: Cell / Method: cryo EM|
|Sample solution||pH: 7.4|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 298 K|
Details: back-side blotting for 1.5-2.5 seconds before plunging.
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.57 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 26000.0 X (calibrated) / Imaging mode: BRIGHT FIELD / Energy filter: GIF Bioquantum|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Processing||Method: subtomogram averaging / Applied symmetry: C1 (asymmetric) / Number of subtomograms: 2333|
|3D reconstruction||Software: IMOD / Resolution: 27 Å / Resolution method: FSC 0.5 CUT-OFF|
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