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- PDB-5ubq: Cryo-EM structure of ciliary microtubule doublet -

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Basic information

Entry
Database: PDB / ID: 5ubq
TitleCryo-EM structure of ciliary microtubule doublet
Components
  • Tubulin alpha chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / cilia / doublet / axoneme / tubulin
Function / homology
Function and homology information


microtubule-based process / structural constituent of cytoskeleton / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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 ...Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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: 5.7 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 21, 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
Revision 1.6Dec 25, 2024Group: Advisory / Data collection ...Advisory / Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature / pdbx_struct_oper_list / pdbx_validate_close_contact / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
C: Tubulin alpha chain
D: Tubulin beta chain
E: Tubulin alpha chain
F: Tubulin beta chain
A: Tubulin alpha chain
B: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)293,36415
Polymers290,3926
Non-polymers2,9729
Water00
1
C: Tubulin alpha chain
D: Tubulin beta chain
E: Tubulin alpha chain
F: Tubulin beta chain
A: Tubulin alpha chain
B: Tubulin beta chain
hetero molecules
x 23


Theoretical massNumber of molelcules
Total (without water)6,747,382345
Polymers6,679,024138
Non-polymers68,357207
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation22
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
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.: 3 / 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.: 3 / Source method: isolated from a natural source / Source: (natural) Tetrahymena thermophila (eukaryote) / References: UniProt: P41352
#3: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 3 / 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.: 3 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
Has protein modificationY

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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: 8nm-repeat tubulin lattice of microtubule doublet / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1-#2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / Strain: SB255 / Cellular location: cilia / Organelle: cilia
Buffer solutionpH: 7.4
SpecimenConc.: 2 mg/ml / Embedding 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 FIELD / 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 real images: 5983
Image scansMovie frames/image: 7 / Used frames/image: 1-7

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Processing

EM software
IDNameVersionCategory
1E2Helix Boxerparticle selection
2EPU1.6image acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
9SPIDER19.02initial Euler assignment
10RELION1.4,2.0final Euler assignment
12RELION1.4,2.03D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 127429
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 127429 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL

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