- EMDB-7805: Subtomogram average (699 axonemal repeats) of isolated Rib72A/B d... -
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Open data
ID or keywords:
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Basic information
Entry
Database: EMDB / ID: EMD-7805
Title
Subtomogram average (699 axonemal repeats) of isolated Rib72A/B double knock out Tetrahymena cilia showing the lumen of the ciliary doublet microtubule (DMT)
Map data
Rib72A/BKO Tetrahymena
Sample
Organelle or cellular component: Subtomogram average (699 axonemal repeats) of isolated Rib72A/B double knock out Tetrahymena cilia showing the lumen of the ciliary doublet microtubule (DMT)
Biological species
Tetrahymena thermophila (eukaryote)
Method
subtomogram averaging / cryo EM / Resolution: 40.1 Å
Journal: 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
Deposition
Apr 20, 2018
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Header (metadata) release
May 16, 2018
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Map release
Sep 5, 2018
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Update
Oct 24, 2018
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Current status
Oct 24, 2018
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_7805.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Rib72A/BKO Tetrahymena
Voxel size
X=Y=Z: 10.77 Å
Density
Contour Level
By AUTHOR: 126. / Movie #1: 126
Minimum - Maximum
63.828963999999999 - 188.034789999999987
Average (Standard dev.)
117.415229999999994 (±12.210169)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
100
100
100
Spacing
100
100
100
Cell
A=B=C: 1077.0 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
10.77
10.77
10.77
M x/y/z
100
100
100
origin x/y/z
0.000
0.000
0.000
length x/y/z
1077.000
1077.000
1077.000
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
100
100
100
D min/max/mean
63.829
188.035
117.415
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Supplemental data
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Sample components
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Entire : Subtomogram average (699 axonemal repeats) of isolated Rib72A/B d...
Entire
Name: Subtomogram average (699 axonemal repeats) of isolated Rib72A/B double knock out Tetrahymena cilia showing the lumen of the ciliary doublet microtubule (DMT)
Components
Organelle or cellular component: Subtomogram average (699 axonemal repeats) of isolated Rib72A/B double knock out Tetrahymena cilia showing the lumen of the ciliary doublet microtubule (DMT)
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Supramolecule #1: Subtomogram average (699 axonemal repeats) of isolated Rib72A/B d...
Supramolecule
Name: Subtomogram average (699 axonemal repeats) of isolated Rib72A/B double 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
Method
cryo EM
Processing
subtomogram averaging
Aggregation state
tissue
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Sample preparation
Buffer
pH: 7.4 Component:
Concentration
Formula
Name
30.0 mM
C8H18N2O4S
HEPES
25.0 mM
KCL
potassium chloride
5.0 mM
MgSO4
magnesium sulfate
1.0 mM
EGTA
0.1 mM
EDTA
Details: Solutions were freshly made before use
Grid
Model: 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
Vitrification
Cryogen 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
Microscope
FEI TECNAI F30
Specialist optics
Energy filter - Name: GIF 2000 / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recording
Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Average electron dose: 1.5 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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