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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 / homology
Function and homology information


microtubule-based process / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain ...Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Tubulin alpha chain / Tubulin beta chain
Similarity search - Component
Biological speciesTetrahymena thermophila (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsIchikawa, M. / Liu, D. / Kastritis, P.L. / Basu, K. / Bui, K.H.
Funding support Canada, 1items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)69462 Canada
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.
History
DepositionDec 22, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 10, 2017Provider: repository / Type: Initial release
Revision 1.1May 17, 2017Group: Database references
Revision 1.2Sep 27, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.3May 16, 2018Group: Data collection / Derived calculations
Category: pdbx_point_symmetry / pdbx_struct_assembly ...pdbx_point_symmetry / pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_oper_list
Revision 1.4Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.5Jan 15, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

Movie
  • Biological unit as complete point assembly
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-8539
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  • Superimposition on EM map
  • EMDB-8539
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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
Polymers96,7972
Non-polymers9913
Water0
1
A: Tubulin alpha chain
B: Tubulin beta chain
hetero molecules

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

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


Theoretical massNumber of molelcules
Total (without water)293,36415
Polymers290,3926
Non-polymers2,9729
Water0
TypeNameSymmetry operationNumber
point symmetry operation3
2


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
SymmetryPoint symmetry: (Schoenflies symbol: C1 (asymmetric))

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Components

#1: Protein Tubulin alpha chain / Alpha-tubulin


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


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


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Protofilament from ciliary microtubule doubletMicrotubule
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1-#2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / 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 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: Blot force 3 for 5 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Nominal defocus max: 3800 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / 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) / Num. of grids imaged: 4 / Num. of real images: 5983
Image scansWidth: 4096 / 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
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10MATLABinitial Euler assignment
11AV3final Euler assignment
13AV33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 127428
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. 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 buildingProtocol: BACKBONE TRACE / Space: REAL / Target criteria: minimization global

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