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- EMDB-6117: A molecular ruler determines the repeat length in eukaryotic cili... -

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Basic information

Entry
Database: EMDB / ID: EMD-6117
TitleA molecular ruler determines the repeat length in eukaryotic cilia and flagella
Map dataFAP59-EL3/FAP172-EL3 axoneme
Sample
  • Sample: FAP59-EL3/FAP172-EL3 axoneme
  • Organelle or cellular component: axoneme
Keywordscryo-electron tomography / cilia and flagella / molecular ruler / FAP59 / FAP172 / CCDC39 / CCDC40
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 52.0 Å
AuthorsOda T / Yanagisawa H / Kamiya R / Kikkawa M
CitationJournal: Science / Year: 2014
Title: A molecular ruler determines the repeat length in eukaryotic cilia and flagella.
Authors: Toshiyuki Oda / Haruaki Yanagisawa / Ritsu Kamiya / Masahide Kikkawa /
Abstract: Existence of cellular structures with specific size raises a fundamental question in biology: How do cells measure length? One conceptual answer to this question is by a molecular ruler, but examples ...Existence of cellular structures with specific size raises a fundamental question in biology: How do cells measure length? One conceptual answer to this question is by a molecular ruler, but examples of such rulers in eukaryotes are lacking. In this work, we identified a molecular ruler in eukaryotic cilia and flagella. Using cryo-electron tomography, we found that FAP59 and FAP172 form a 96-nanometer (nm)-long complex in Chlamydomonas flagella and that the absence of the complex disrupted 96-nm repeats of axonemes. Furthermore, lengthening of the FAP59/172 complex by domain duplication resulted in extension of the repeats up to 128 nm, as well as duplication of specific axonemal components. Thus, the FAP59/172 complex is the molecular ruler that determines the 96-nm repeat length and arrangements of components in cilia and flagella.
History
DepositionSep 30, 2014-
Header (metadata) releaseOct 15, 2014-
Map releaseNov 26, 2014-
UpdateJun 17, 2015-
Current statusJun 17, 2015Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 124
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 124
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_6117.map.gz / Format: CCP4 / Size: 48.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFAP59-EL3/FAP172-EL3 axoneme
Voxel sizeX=Y=Z: 6.07 Å
Density
Contour LevelBy AUTHOR: 124.0 / Movie #1: 124
Minimum - Maximum0.0 - 250.685714719999993
Average (Standard dev.)43.694305419999999 (±57.863956450000003)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400180180
Spacing400180180
CellA: 1092.6 Å / B: 2428.0 Å / C: 1092.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.076.076.07
M x/y/z180400180
origin x/y/z0.0000.0000.000
length x/y/z1092.6002428.0001092.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-24-24-24
NX/NY/NZ494949
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180400180
D min/max/mean0.000250.68643.694

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Supplemental data

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Sample components

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Entire : FAP59-EL3/FAP172-EL3 axoneme

EntireName: FAP59-EL3/FAP172-EL3 axoneme
Components
  • Sample: FAP59-EL3/FAP172-EL3 axoneme
  • Organelle or cellular component: axoneme

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Supramolecule #1000: FAP59-EL3/FAP172-EL3 axoneme

SupramoleculeName: FAP59-EL3/FAP172-EL3 axoneme / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: axoneme

SupramoleculeName: axoneme / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Organelle: Flagella

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.2
Details: 30 mM Hepes-NaOH, 5 mM MgCl2, 1 mM DTT, 1 mM EGTA, 50 mM NaCl
GridDetails: 300 mesh copper grid with holey carbon support, glow discharged in the air
VitrificationCryogen name: ETHANE / Chamber humidity: 98 % / Chamber temperature: 93 K / Instrument: LEICA EM GP / Method: Blot for 5 seconds before plunging

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Electron microscopy

MicroscopeJEOL 3100FFC
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 7.0 µm / Nominal defocus min: 6.0 µm / Nominal magnification: 25700
Specialist opticsEnergy filter - Name: Omega / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -65 ° / Tilt series - Axis1 - Max angle: 65 °
DateJun 26, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 100 e/Å2

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 52.0 Å / Resolution method: OTHER
DetailsNumber of tilts (projections) used in 3D reconstruction: 65 Tomographic tilt angle increment: 2

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