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- EMDB-10900: cryo-ET/ subtomogram averaging of primary cilia reveals actin fil... -

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Basic information

Entry
Database: EMDB / ID: EMD-10900
Titlecryo-ET/ subtomogram averaging of primary cilia reveals actin filaments in the axoneme
Map datacryo-subtomogram averaging of primary cilia actin filaments
Sample
  • Complex: Cryo-subtomogram averaging of actin filaments in primary cilia
Biological speciesCanis lupus familiaris (dog)
Methodsubtomogram averaging / cryo EM / Resolution: 45.0 Å
AuthorsKiesel P / Alvarez Viar G / Tsoy N / Maraspini R / Honigmann A / Pigino G
Funding support1 items
OrganizationGrant numberCountry
European Research Council (ERC)819826
Citation
Journal: Nat Struct Mol Biol / Year: 2020
Title: The molecular structure of mammalian primary cilia revealed by cryo-electron tomography.
Authors: Petra Kiesel / Gonzalo Alvarez Viar / Nikolai Tsoy / Riccardo Maraspini / Peter Gorilak / Vladimir Varga / Alf Honigmann / Gaia Pigino /
Abstract: Primary cilia are microtubule-based organelles that are important for signaling and sensing in eukaryotic cells. Unlike the thoroughly studied motile cilia, the three-dimensional architecture and ...Primary cilia are microtubule-based organelles that are important for signaling and sensing in eukaryotic cells. Unlike the thoroughly studied motile cilia, the three-dimensional architecture and molecular composition of primary cilia are largely unexplored. Yet, studying these aspects is necessary to understand how primary cilia function in health and disease. We developed an enabling method for investigating the structure of primary cilia isolated from MDCK-II cells at molecular resolution by cryo-electron tomography. We show that the textbook '9 + 0' arrangement of microtubule doublets is only present at the primary cilium base. A few microns out, the architecture changes into an unstructured bundle of EB1-decorated microtubules and actin filaments, putting an end to a long debate on the presence or absence of actin filaments in primary cilia. Our work provides a plethora of insights into the molecular structure of primary cilia and offers a methodological framework to study these important organelles.
#1: Journal: Biorxiv / Year: 2020
Title: The molecular structure of primary cilia revealed by cryo-electron tomography
Authors: Kiesel P / Alvarez Viar G / Tsoy N / Maraspini R / Honigmann A / Pigino G
History
DepositionApr 22, 2020-
Header (metadata) releaseSep 9, 2020-
Map releaseSep 9, 2020-
UpdateMay 19, 2021-
Current statusMay 19, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.497
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.497
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10900.png

Downloads & links

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Map

FileDownload / File: emd_10900.map.gz / Format: CCP4 / Size: 26.4 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-subtomogram averaging of primary cilia actin filaments
Voxel sizeX=Y=Z: 9.422 Å
Density
Contour LevelBy AUTHOR: 0.497 / Movie #1: 0.497
Minimum - Maximum-3.0304651 - 3.461957
Average (Standard dev.)-0.1655966 (±1.0477972)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin121417
Dimensions431312
Spacing134312
CellA: 122.486 Å / B: 405.146 Å / C: 113.063995 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z9.4229.4229.422
M x/y/z134312
origin x/y/z0.0000.0000.000
length x/y/z122.486405.146113.064
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS141217
NC/NR/NS134312
D min/max/mean-3.0303.462-0.166

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Supplemental data

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Sample components

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Entire : Cryo-subtomogram averaging of actin filaments in primary cilia

EntireName: Cryo-subtomogram averaging of actin filaments in primary cilia
Components
  • Complex: Cryo-subtomogram averaging of actin filaments in primary cilia

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Supramolecule #1: Cryo-subtomogram averaging of actin filaments in primary cilia

SupramoleculeName: Cryo-subtomogram averaging of actin filaments in primary cilia
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Canis lupus familiaris (dog) / Organ: Kidney / Tissue: tubule epithelial tissue / Organelle: primary cilium / Location in cell: axoneme

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.25
GridModel: Quantifoil R3.5/1 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 1.6 e/Å2

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Image processing

Crystal parametersUnit cell - A: 1 Å / Unit cell - B: 1 Å / Unit cell - C: 1 Å / Unit cell - γ: 1 ° / Unit cell - α: 1 ° / Unit cell - β: 1 ° / Space group: 1
ExtractionNumber tomograms: 4 / Number images used: 129
CTF correctionSoftware - Name: CTFPHASEFLIP
Final angle assignmentType: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 45.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PEET
Details: The diffraction pattern show a layerline at 57.4 Angstrom, which represent the distance between actin monomers.
Number subtomograms used: 129

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