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- EMDB-6756: Electrostatic interaction between polyglutamylated tubulin and th... -

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Basic information

Entry
Database: EMDB / ID: EMD-6756
TitleElectrostatic interaction between polyglutamylated tubulin and the nexin-dynein regulatory complex regulates flagellar motility
Map dataDMT structure of wild type axoneme
Sample
  • Organelle or cellular component: Outer doublet microtubule of pf2fp3 axoneme labeled with polyE2 Fab fragments
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 50.0 Å
AuthorsKubo T / Oda T
CitationJournal: Mol Biol Cell / Year: 2017
Title: Electrostatic interaction between polyglutamylated tubulin and the nexin-dynein regulatory complex regulates flagellar motility.
Authors: Tomohiro Kubo / Toshiyuki Oda /
Abstract: Tubulins undergo various posttranslational modifications. Among them, polyglutamylation is involved in the motility of eukaryotic flagella and the stability of the axonemal microtubules. However, it ...Tubulins undergo various posttranslational modifications. Among them, polyglutamylation is involved in the motility of eukaryotic flagella and the stability of the axonemal microtubules. However, it remains unclear where polyglutamylated tubulin localizes precisely within the axoneme and how tubulin polyglutamylation affects flagellar motility. In this study, we identified the three-dimensional localization of the polyglutamylated tubulin in flagella using antibody labeling and cryo-electron tomography. Polyglutamylated tubulins specifically located in close proximity to a microtubule-cross-bridging structure called the nexin-dynein regulatory complex (N-DRC). Because N-DRC is positively charged, we hypothesized that there is an electrostatic interaction between the polyglutamylated tubulin and the N-DRC, and therefore we mutated the amino acid sequences of DRC4 to modify the charge of the N-DRC. We found that both augmentation and reduction of the positive charge on DRC4 resulted in reduced flagellar motility Moreover, reduced motility in a mutant with a structurally defective N-DRC was partially restored by increasing the positive charge on DRC4. These results clearly indicate that beating motion of flagella is maintained by the electrostatic cross-bridge formed between the negatively charged polyglutamylated tubulins and the positively charged N-DRC.
History
DepositionJun 12, 2017-
Header (metadata) releaseJul 12, 2017-
Map releaseJul 12, 2017-
UpdateJun 20, 2018-
Current statusJun 20, 2018Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 301
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 301
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6756.png

Downloads & links

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Map

FileDownload / File: emd_6756.map.gz / Format: CCP4 / Size: 11.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDMT structure of wild type axoneme
Voxel sizeX=Y=Z: 7.2 Å
Density
Contour LevelBy AUTHOR: 301. / Movie #1: 301
Minimum - Maximum0. - 556.36749999999995
Average (Standard dev.)202.559830000000005 (±97.670050000000003)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200125120
Spacing125200120
CellA: 900.0 Å / B: 1440.0 Å / C: 864.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.27.27.2
M x/y/z125200120
origin x/y/z0.0000.0000.000
length x/y/z900.0001440.000864.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS125200120
D min/max/mean0.000556.367202.560

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Supplemental data

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Sample components

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Entire : Outer doublet microtubule of pf2fp3 axoneme labeled with polyE2 F...

EntireName: Outer doublet microtubule of pf2fp3 axoneme labeled with polyE2 Fab fragments
Components
  • Organelle or cellular component: Outer doublet microtubule of pf2fp3 axoneme labeled with polyE2 Fab fragments

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Supramolecule #1: Outer doublet microtubule of pf2fp3 axoneme labeled with polyE2 F...

SupramoleculeName: Outer doublet microtubule of pf2fp3 axoneme labeled with polyE2 Fab fragments
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 statefilament

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 7.2
Details: 30 mM Hepes-NaOH pH 7.2, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, 50 mM NaCl
GridModel: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 5.5 seconds before plunging.

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Electron microscopy

MicroscopeJEOL 3100FEF
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Specialist opticsEnergy filter - Name: Omega / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: GATAN 914 HIGH TILT LIQUID NITROGEN CRYO TRANSFER TOMOGRAPHY HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 1.7 e/Å2

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Image processing

ExtractionNumber tomograms: 8 / Number images used: 892 / Software - Name: PEET
CTF correctionSoftware - Name: IMOD
Final angle assignmentType: NOT APPLICABLE / Software - Name: PEET
Final reconstructionResolution.type: BY AUTHOR / Resolution: 50.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PEET / Number subtomograms used: 766
FSC plot (resolution estimation)

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