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- EMDB-24375: B-tubule of axonemal microtubule doublet in 48 nm repeat from Tet... -

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Basic information

Entry
Database: EMDB / ID: EMD-24375
TitleB-tubule of axonemal microtubule doublet in 48 nm repeat from Tetrahymena thermophila mutant fap115 knockout
Map dataB-tubule of axonemal microtubule doublet in 48 nm repeat from Tetrahymena thermophila mutant fap115 knockout
Sample
  • Organelle or cellular component: Axonemal microtubule doublet from Tetrahymena thermophile
Biological speciesTetrahymena thermophila (eukaryote)
Methodsubtomogram averaging / cryo EM / Resolution: 12.0 Å
AuthorsLi S / Fernandez JJ / Fabritius A / Agard DA / Winey M
Funding support United States, Spain, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM127571-04 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R35GM118099-05 United States
Spanish National Research CouncilSAF2017-84565-R Spain
CitationJournal: Life Sci Alliance / Year: 2022
Title: Electron cryo-tomography structure of axonemal doublet microtubule from .
Authors: Sam Li / Jose-Jesus Fernandez / Amy S Fabritius / David A Agard / Mark Winey /
Abstract: Doublet microtubules (DMTs) provide a scaffold for axoneme assembly in motile cilia. Aside from α/β tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMTs, ...Doublet microtubules (DMTs) provide a scaffold for axoneme assembly in motile cilia. Aside from α/β tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMTs, collectively named the microtubule inner proteins (MIPs). We used cryoET to study axoneme DMT isolated from We present the structures of DMT at nanometer and sub-nanometer resolution. The structures confirm that MIP RIB72A/B binds to the luminal wall of DMT by multiple DM10 domains. We found FAP115, an MIP-containing multiple EF-hand domains, located at the interface of four-tubulin dimers in the lumen of A-tubule. It contacts both lateral and longitudinal tubulin interfaces and playing a critical role in DMT stability. We observed substantial structure heterogeneity in DMT in an knockout strain, showing extensive structural defects beyond the FAP115-binding site. The defects propagate along the axoneme. Finally, by comparing DMT structures from and , we have identified a number of conserved MIPs as well as MIPs that are unique to each organism. This conservation and diversity of the DMT structures might be linked to their specific functions. Our work provides structural insights essential for understanding the roles of MIPs during motile cilium assembly and function, as well as their relationships to human ciliopathies.
History
DepositionJul 2, 2021-
Header (metadata) releaseDec 29, 2021-
Map releaseDec 29, 2021-
UpdateJan 19, 2022-
Current statusJan 19, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.221
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.221
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24375.png

Downloads & links

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Map

FileDownload / File: emd_24375.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationB-tubule of axonemal microtubule doublet in 48 nm repeat from Tetrahymena thermophila mutant fap115 knockout
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.65 Å/pix.
x 250 pix.
= 662.5 Å		2.65 Å/pix.
x 250 pix.
= 662.5 Å		2.65 Å/pix.
x 250 pix.
= 662.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.65 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.221 / Movie #1: 0.221
Minimum - Maximum-0.7730423 - 0.820239
Average (Standard dev.)0.001202253 (±0.082663536)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00250
Dimensions250250250
Spacing250250250
CellA=B=C: 662.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.652.652.65
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z662.500662.500662.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00250
NC/NR/NS250250250
D min/max/mean-0.7730.8200.001

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Supplemental data

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Sample components

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Entire : Axonemal microtubule doublet from Tetrahymena thermophile

EntireName: Axonemal microtubule doublet from Tetrahymena thermophile
Components
  • Organelle or cellular component: Axonemal microtubule doublet from Tetrahymena thermophile

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Supramolecule #1: Axonemal microtubule doublet from Tetrahymena thermophile

SupramoleculeName: Axonemal microtubule doublet from Tetrahymena thermophile
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / Organelle: Flagellum / Location in cell: Cell Surface

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 70.0 K / Max: 70.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 0.36 sec. / Average electron dose: 1.31 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 4.0 µm / Calibrated defocus min: 2.0 µm / Calibrated magnification: 18868 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 33000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 9502
ExtractionNumber tomograms: 64 / Number images used: 9502
Final angle assignmentType: MAXIMUM LIKELIHOOD

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