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- EMDB-7806: Subtomogram average (758 axonemal repeats) of isolated Rib72A kno... -

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Basic information

Entry
Database: EMDB / ID: EMD-7806
TitleSubtomogram average (758 axonemal repeats) of isolated Rib72A knock out Tetrahymena cilia showing the lumen of the ciliary doublet microtubule (DMT)
Map dataRib72AKO
Sample
  • Organelle or cellular component: Subtomogram average (758 axonemal repeats) of isolated Rib72A knock out Tetrahymena cilia showing the lumen of the ciliary doublet microtubule (DMT)
Biological speciesTetrahymena thermophila (eukaryote)
Methodsubtomogram averaging / cryo EM / Resolution: 40.1 Å
AuthorsStoddard D / Zhao Y / Bayless B / Gui L / Louka P / Dave D / Surwayanshi S / Tomasi R / Dupuis-Williams P / Baroud C ...Stoddard D / Zhao Y / Bayless B / Gui L / Louka P / Dave D / Surwayanshi S / Tomasi R / Dupuis-Williams P / Baroud C / Gaertig J / Winey M / Nicastro D
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR01GM083122 United States
CitationJournal: Mol Biol Cell / Year: 2018
Title: Tetrahymena RIB72A and RIB72B are microtubule inner proteins in the ciliary doublet microtubules.
Authors: Daniel Stoddard / Ying Zhao / Brian A Bayless / Long Gui / Panagiota Louka / Drashti Dave / Swati Suryawanshi / Raphaël F-X Tomasi / Pascale Dupuis-Williams / Charles N Baroud / Jacek ...Authors: Daniel Stoddard / Ying Zhao / Brian A Bayless / Long Gui / Panagiota Louka / Drashti Dave / Swati Suryawanshi / Raphaël F-X Tomasi / Pascale Dupuis-Williams / Charles N Baroud / Jacek Gaertig / Mark Winey / Daniela Nicastro /
Abstract: Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia, and flagella. In contrast to dynamic cytoplasmic micro-tubules, their luminal ...Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia, and flagella. In contrast to dynamic cytoplasmic micro-tubules, their luminal surface is coated with regularly arranged microtubule inner proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical, and imaging techniques, we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins colocalize to Tetrahymena cilia and basal bodies but assemble independently. Cryoelectron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4, and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility.
History
DepositionApr 20, 2018-
Header (metadata) releaseMay 16, 2018-
Map releaseSep 5, 2018-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 126
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 126
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7806.png

Downloads & links

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Map

FileDownload / File: emd_7806.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRib72AKO
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10.77 Å/pix.
x 100 pix.
= 1077. Å		10.77 Å/pix.
x 100 pix.
= 1077. Å		10.77 Å/pix.
x 100 pix.
= 1077. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 10.77 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 126 / Movie #1: 126
Minimum - Maximum76.79396 - 172.50739
Average (Standard dev.)116.80304 (±9.595288)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 1077.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z10.7710.7710.77
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z1077.0001077.0001077.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean76.794172.507116.803

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Supplemental data

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Sample components

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Entire : Subtomogram average (758 axonemal repeats) of isolated Rib72A kno...

EntireName: Subtomogram average (758 axonemal repeats) of isolated Rib72A knock out Tetrahymena cilia showing the lumen of the ciliary doublet microtubule (DMT)
Components
  • Organelle or cellular component: Subtomogram average (758 axonemal repeats) of isolated Rib72A knock out Tetrahymena cilia showing the lumen of the ciliary doublet microtubule (DMT)

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Supramolecule #1: Subtomogram average (758 axonemal repeats) of isolated Rib72A kno...

SupramoleculeName: Subtomogram average (758 axonemal repeats) of isolated Rib72A knock out Tetrahymena cilia showing the lumen of the ciliary doublet microtubule (DMT)
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / Strain: Rib72A/B KO

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statetissue

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationFormulaName
30.0 mMC8H18N2O4SHEPES
25.0 mMKCLpotassium chloride
5.0 mMMgSO4magnesium sulfate
1.0 mMEGTA
0.1 mMEDTA

Details: Solutions were freshly made before use
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER
Details: back-side blotting for 1.5-2.5 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI F30
Specialist opticsEnergy filter - Name: GIF 2000 / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recordingFilm or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Average electron dose: 1.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 8.0 µm / Calibrated magnification: 13500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus min: 6.0 µm
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 40.1 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMOD / Number subtomograms used: 758
ExtractionNumber tomograms: 5 / Number images used: 758 / Software - Name: MATLAB
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: PEET

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