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- PDB-6ve7: The inner junction complex of Chlamydomonas reinhardtii doublet m... -

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Basic information

Entry
Database: PDB / ID: 6ve7
TitleThe inner junction complex of Chlamydomonas reinhardtii doublet microtubule
Components
  • Cilia- and flagella-associated protein 20
  • FAP276
  • FAP52
  • Flagellar associated protein
  • PACRG
  • Protein Flattop homolog
  • Tubulin alpha
  • Tubulin beta
KeywordsSTRUCTURAL PROTEIN / cilia / doublet / axoneme / microtubule inner protein
Function / homology
Function and homology information


axonemal central pair / axonemal outer doublet / membrane-bounded organelle / positive regulation of cilium-dependent cell motility / regulation of cilium beat frequency involved in ciliary motility / establishment of protein localization to organelle / axoneme assembly / axonemal microtubule / intracellular organelle / motile cilium ...axonemal central pair / axonemal outer doublet / membrane-bounded organelle / positive regulation of cilium-dependent cell motility / regulation of cilium beat frequency involved in ciliary motility / establishment of protein localization to organelle / axoneme assembly / axonemal microtubule / intracellular organelle / motile cilium / microtubule associated complex / cilium assembly / microtubule-based process / Hsp70 protein binding / ciliary basal body / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Hsp90 protein binding / structural constituent of cytoskeleton / microtubule / cytoskeleton / calmodulin binding / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Protein Flattop / Enkurin domain / Calmodulin-binding / Enkurin domain profile. / CFA20 domain / Cilia- and flagella-associated protein 20/CFAP20DC / CFA20 domain / Parkin co-regulated protein / Parkin co-regulated protein / Tubulin-beta mRNA autoregulation signal. ...Protein Flattop / Enkurin domain / Calmodulin-binding / Enkurin domain profile. / CFA20 domain / Cilia- and flagella-associated protein 20/CFAP20DC / CFA20 domain / Parkin co-regulated protein / Parkin co-regulated protein / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily / Armadillo-type fold / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / Cilia- and flagella-associated protein 52 / Flagellar associated protein / Cilia- and flagella-associated protein 20 / Protein Flattop homolog / Uncharacterized protein / Parkin-co-regulated gene product / Tubulin beta-1/beta-2 chain / Tubulin alpha-1 chain
Similarity search - Component
Biological speciesChlamydomonas reinhardtii (plant)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsKhalifa, A.A.Z. / Ichikawa, M. / Bui, K.H.
Funding support Canada, 3items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2016-04954 Canada
Canadian Institutes of Health Research (CIHR)PJT-156354 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2018-04813 Canada
CitationJournal: Elife / Year: 2020
Title: The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications.
Authors: Ahmad Abdelzaher Zaki Khalifa / Muneyoshi Ichikawa / Daniel Dai / Shintaroh Kubo / Corbin Steven Black / Katya Peri / Thomas S McAlear / Simon Veyron / Shun Kai Yang / Javier Vargas / ...Authors: Ahmad Abdelzaher Zaki Khalifa / Muneyoshi Ichikawa / Daniel Dai / Shintaroh Kubo / Corbin Steven Black / Katya Peri / Thomas S McAlear / Simon Veyron / Shun Kai Yang / Javier Vargas / Susanne Bechstedt / Jean-François Trempe / Khanh Huy Bui /
Abstract: Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and β-tubulin heterodimers, form protofilaments that associate ...Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and β-tubulin heterodimers, form protofilaments that associate laterally into the hollow microtubule. Microtubule also exists as highly stable doublet microtubules in the cilia where stability is needed for ciliary beating and function. The doublet microtubule maintains its stability through interactions at its inner and outer junctions where its A- and B-tubules meet. Here, using cryo-electron microscopy, bioinformatics and mass spectrometry of the doublets of and , we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction-associated protein, FAP126, thus presenting the complete answer to the inner junction identity and localization. Our structural study of the doublets shows that the inner junction serves as an interaction hub that involves tubulin post-translational modifications. These interactions contribute to the stability of the doublet and hence, normal ciliary motility.
History
DepositionDec 30, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 5, 2020Provider: repository / Type: Initial release

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Structure visualization

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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Flagellar associated protein
B: FAP52
C: FAP52
D: Tubulin alpha
E: Tubulin alpha
F: Tubulin alpha
G: Tubulin alpha
H: Protein Flattop homolog
I: Tubulin beta
J: Tubulin beta
K: Tubulin beta
L: Tubulin alpha
M: Tubulin alpha
N: Tubulin beta
O: Tubulin beta
P: Tubulin alpha
Q: Tubulin beta
R: Tubulin beta
S: Tubulin alpha
T: Tubulin beta
U: Tubulin beta
V: Tubulin beta
W: FAP276
X: Tubulin alpha
Y: Tubulin alpha
Z: Tubulin alpha
a: Tubulin beta
b: Tubulin beta
c: Cilia- and flagella-associated protein 20
d: PACRG
e: Tubulin alpha
f: Tubulin alpha
g: Cilia- and flagella-associated protein 20
h: Tubulin alpha
i: Tubulin beta
j: Tubulin beta
k: Tubulin alpha
l: PACRG
m: Tubulin alpha
n: PACRG
o: Tubulin alpha
p: Tubulin beta
q: Tubulin beta
r: Tubulin beta
s: Tubulin alpha
t: Tubulin beta
u: Tubulin beta
v: Tubulin beta
w: Tubulin beta
x: FAP276
y: Tubulin alpha
z: Cilia- and flagella-associated protein 20
0: Tubulin alpha
1: Tubulin alpha
2: PACRG
3: Cilia- and flagella-associated protein 20
4: Tubulin beta
5: Tubulin alpha
6: Tubulin alpha
7: Tubulin alpha
8: Tubulin beta
9: Tubulin beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,845,886150
Polymers2,808,44762
Non-polymers37,43988
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 8 types, 62 molecules ABCDEFGLMPSXYZefhkmosy01567HIJ...

#1: Protein Flagellar associated protein


Mass: 27019.803 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Chlamydomonas reinhardtii (plant) / References: UniProt: A8J098
#2: Protein FAP52


Mass: 68546.508 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Chlamydomonas reinhardtii (plant) / References: UniProt: A0A2K3D260
#3: Protein ...
Tubulin alpha / Tubulin alpha-1 chain


Mass: 49638.008 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Source: (natural) Chlamydomonas reinhardtii (plant) / References: UniProt: P09204
#4: Protein Protein Flattop homolog / Cilia- and flagella-associated protein 126 / Flagellum-associated protein 126


Mass: 15435.397 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Chlamydomonas reinhardtii (plant) / References: UniProt: A8IVJ1
#5: Protein ...
Tubulin beta / Tubulin beta-1/beta-2 chain / Beta-tubulin


Mass: 49665.809 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Source: (natural) Chlamydomonas reinhardtii (plant) / References: UniProt: P04690
#6: Protein FAP276


Mass: 9986.308 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Chlamydomonas reinhardtii (plant) / References: UniProt: A0A2K3DTN6
#7: Protein
Cilia- and flagella-associated protein 20 / Basal body up-regulated protein 22 / Bug22p / Flagellar-associated protein 20


Mass: 22193.566 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Chlamydomonas reinhardtii (plant) / References: UniProt: A8IU92
#8: Protein
PACRG / Parkin-co-regulated gene product


Mass: 34215.148 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Chlamydomonas reinhardtii (plant) / References: UniProt: B1B601

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Non-polymers , 4 types, 88 molecules

#9: Chemical...
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#10: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: Mg
#11: Chemical...
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#12: Chemical
ChemComp-TA1 / TAXOL / Paclitaxel


Mass: 853.906 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: The inner junction complex of Chlamydomonas axoneme / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1-#8 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: CC-124 / Organelle: cilia
Buffer solutionpH: 7.4
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 4000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 45 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 9528
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096 / Movie frames/image: 7 / Used frames/image: 1-7

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Processing

EM software
IDNameVersionCategoryDetails
1EMAN22.05particle selectione2helixboxer.py
2EPU1.5image acquisition
4Gctf1CTF correction
7UCSF Chimera1.4model fitting
9PHENIX9.03model refinement
10RELION3.0binitial Euler assignment
11RELION3final Euler assignment
12RELION3.0bclassification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 270713
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 270713 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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