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- PDB-5ucy: Cryo-EM map of protofilament of microtubule doublet -

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Basic information

Entry
Database: PDB / ID: 5ucy
TitleCryo-EM map of protofilament of microtubule doublet
Components
  • Tubulin alpha chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / cilia / doublet / protofilament / tubulin
Function/homologyBeta tubulin, autoregulation binding site / Tubulin-beta mRNA autoregulation signal. / Beta tubulin / Alpha tubulin / Tubulin / Tubulin, C-terminal / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / microtubule-based process / Tubulin/FtsZ, C-terminal domain superfamily ...Beta tubulin, autoregulation binding site / Tubulin-beta mRNA autoregulation signal. / Beta tubulin / Alpha tubulin / Tubulin / Tubulin, C-terminal / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / microtubule-based process / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily / structural constituent of cytoskeleton / Tubulin C-terminal domain / Tubulin/FtsZ family, GTPase domain / microtubule / GTPase activity / GTP binding / cytoplasm / Tubulin alpha chain / Tubulin beta chain
Function and homology information
Specimen sourceTetrahymena thermophila / Tetrahymena / eukaryote
MethodElectron microscopy (4.6 Å resolution / Filament / Single particle) / Transmission electron microscopy
AuthorsIchikawa, M. / Liu, D. / Kastritis, P.L. / Basu, K. / Bui, K.H.
CitationJournal: Nat Commun / Year: 2017
Title: Subnanometre-resolution structure of the doublet microtubule reveals new classes of microtubule-associated proteins.
Authors: Muneyoshi Ichikawa / Dinan Liu / Panagiotis L Kastritis / Kaustuv Basu / Tzu Chin Hsu / Shunkai Yang / Khanh Huy Bui
Abstract: Cilia are ubiquitous, hair-like appendages found in eukaryotic cells that carry out functions of cell motility and sensory reception. Cilia contain an intriguing cytoskeletal structure, termed the ...Cilia are ubiquitous, hair-like appendages found in eukaryotic cells that carry out functions of cell motility and sensory reception. Cilia contain an intriguing cytoskeletal structure, termed the axoneme that consists of nine doublet microtubules radially interlinked and longitudinally organized in multiple specific repeat units. Little is known, however, about how the axoneme allows cilia to be both actively bendable and sturdy or how it is assembled. To answer these questions, we used cryo-electron microscopy to structurally analyse several of the repeating units of the doublet at sub-nanometre resolution. This structural detail enables us to unambiguously assign α- and β-tubulins in the doublet microtubule lattice. Our study demonstrates the existence of an inner sheath composed of different kinds of microtubule inner proteins inside the doublet that likely stabilizes the structure and facilitates the specific building of the B-tubule.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 22, 2016 / Release: May 10, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0May 10, 2017Structure modelrepositoryInitial release
1.1May 17, 2017Structure modelDatabase references
1.2Sep 27, 2017Structure modelAuthor supporting evidence / Data collectionem_software / pdbx_audit_support_em_software.name / _pdbx_audit_support.funding_organization

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • EMDB-8539
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Assembly

Deposited unit
A: Tubulin alpha chain
B: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,7885
Polyers96,7972
Non-polymers9913
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide Tubulin alpha chain / Alpha-tubulin


Mass: 48788.254 Da / Num. of mol.: 1
Source: (natural) Tetrahymena thermophila / Tetrahymena / eukaryote
References: UniProt:P41351
#2: Protein/peptide Tubulin beta chain / Beta-tubulin


Mass: 48009.199 Da / Num. of mol.: 1
Source: (natural) Tetrahymena thermophila / Tetrahymena / eukaryote
References: UniProt:P41352
#3: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Formula: C10H16N5O14P3 / : Guanosine triphosphate / Comment: GTP (energy-carrying molecule) *YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Formula: Mg / : Magnesium
#5: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Mass: 443.201 Da / Num. of mol.: 1 / Formula: C10H15N5O11P2 / : Guanosine diphosphate / Comment: GDP (energy-carrying molecule) *YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Protofilament from ciliary microtubule doublet / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: 1, 2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Tetrahymena thermophila / Strain: SB255
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 kelvins / Details: Blot force 3 for 5 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 59000 / Nominal defocus max: 3800 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.4 sec. / Electron dose: 45 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Number of grids imaged: 4 / Number of real images: 5983
Image scansDimension width: 4096 / Dimension height: 4096 / Movie frames/image: 7 / Used frames/image: 1-7

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Processing

EM software
IDNameVersionCategory
1E2helixboxer2PARTICLE SELECTION
2EPU1.6IMAGE ACQUISITION
4ctffind4CTF CORRECTION
7ChimeraMODEL FITTING
9PHENIXMODEL REFINEMENT
10MATLABINITIAL EULER ASSIGNMENT
11Tom Av3FINAL EULER ASSIGNMENT
13Tom AV3RECONSTRUCTION
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 127428
SymmetryPoint symmetry: C1
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 23
Details: 23 boxes of the protofilament are boxed out from the doublet map (EMD-8528), aligned and averaged using subtomogram averaging software.
Symmetry type: POINT
Atomic model buildingRef protocol: BACKBONE TRACE / Ref space: REAL / Target criteria: minimization global

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