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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
DescriptorTubulin alpha chain, Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / cilia / doublet / protofilament / tubulin
Specimen sourceTetrahymena thermophila / eukaryote
MethodElectron microscopy (4.6 Å resolution / Filament / Single particle)
AuthorsIchikawa, M. / Liu, D. / Kastritis, P.L. / Basu, K. / Bui, K.H.
CitationNat Commun, 2017, 8, 15035-15035

Nat Commun, 2017, 8, 15035-15035 StrPapers
Subnanometre-resolution structure of the doublet microtubule reveals new classes of microtubule-associated proteins.
Muneyoshi Ichikawa / Dinan Liu / Panagiotis L Kastritis / Kaustuv Basu / Tzu Chin Hsu / Shunkai Yang / Khanh Huy Bui

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 22, 2016 / Release: May 10, 2017
RevisionDateData content typeGroupProviderType
1.0May 10, 2017Structure modelrepositoryInitial release
1.1May 17, 2017Structure modelDatabase references

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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: Polypeptide(L)Tubulin alpha chain / Alpha-tubulin


Mass: 48788.254 Da / Num. of mol.: 1 / Source: (natural) Tetrahymena thermophila / eukaryote / References: UniProt: P41351

Cellular component

Molecular function

Biological process

#2: Polypeptide(L)Tubulin beta chain / Beta-tubulin


Mass: 48009.199 Da / Num. of mol.: 1 / Source: (natural) Tetrahymena thermophila / eukaryote / References: UniProt: P41352

Cellular component

Molecular function

Biological process

#3: ChemicalChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / GTP *YM


Mass: 523.180 Da / Num. of mol.: 1 / Formula: C10H16N5O14P3
#4: ChemicalChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Formula: Mg
#5: ChemicalChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / GDP *YM


Mass: 443.201 Da / Num. of mol.: 1 / Formula: C10H15N5O11P2

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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
IDNameVersionCategoryImage processing IDImaging IDFitting ID
1E2helixboxer2PARTICLE SELECTION1
2EPU1.6IMAGE ACQUISITION1
4ctffind4CTF CORRECTION1
7ChimeraMODEL FITTING1
9PhenixMODEL REFINEMENT1
10MatlabINITIAL EULER ASSIGNMENT1
11Tom Av3FINAL EULER ASSIGNMENT1
13Tom AV3RECONSTRUCTION1
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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