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- EMDB-10935: Pinhead of the microtubule triplet of isolated Trichonympha agili... -

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Basic information

Entry
Database: EMDB / ID: EMD-10935
TitlePinhead of the microtubule triplet of isolated Trichonympha agilis centriole
Map data
Sample
  • Organelle or cellular component: Isolated basal body from Trichonympha agilis
Biological speciesTrichonympha agilis (eukaryote)
Methodsubtomogram averaging / cryo EM / Resolution: 39.0 Å
AuthorsNazarov S
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)AdG 340227 Switzerland
CitationJournal: EMBO J / Year: 2020
Title: Novel features of centriole polarity and cartwheel stacking revealed by cryo-tomography.
Authors: Sergey Nazarov / Alexandra Bezler / Georgios N Hatzopoulos / Veronika Nemčíková Villímová / Davide Demurtas / Maeva Le Guennec / Paul Guichard / Pierre Gönczy /
Abstract: Centrioles are polarized microtubule-based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers of SAS-6 proteins. A cryo-tomography map ...Centrioles are polarized microtubule-based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers of SAS-6 proteins. A cryo-tomography map of centrioles from the termite flagellate Trichonympha spp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub-tomogram averaging (STA) in T. spp. and Trichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead, and A-C linker. Moreover, we report ~25 Å resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore, STA of centrioles from the distant flagellate Teranympha mirabilis uncovers similar cartwheel architecture and a distinct filamentous CID. Fitting the CrSAS-6 crystal structure into the flagellate maps and analyzing cartwheels generated in vitro indicate that SAS-6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improved STA maps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms.
History
DepositionApr 27, 2020-
Header (metadata) releaseNov 11, 2020-
Map releaseNov 11, 2020-
UpdateMay 26, 2021-
Current statusMay 26, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10935.png

Downloads & links

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Map

FileDownload / File: emd_10935.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 6.982 Å
Density
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-0.33042017 - 0.5108445
Average (Standard dev.)0.0049738595 (±0.06951118)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 558.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.9826.9826.982
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z558.560558.560558.560
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.3300.5110.005

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Supplemental data

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Sample components

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Entire : Isolated basal body from Trichonympha agilis

EntireName: Isolated basal body from Trichonympha agilis
Components
  • Organelle or cellular component: Isolated basal body from Trichonympha agilis

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Supramolecule #1: Isolated basal body from Trichonympha agilis

SupramoleculeName: Isolated basal body from Trichonympha agilis / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Trichonympha agilis (eukaryote)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

BufferpH: 6.8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 2.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 18 / Number images used: 3850
CTF correctionSoftware: (Name: RELION (ver. 3.1), IMOD (ver. 4.9))
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 39.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number subtomograms used: 714
FSC plot (resolution estimation)

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