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- PDB-3jao: Ciliary microtubule doublet -

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Basic information

Entry
Database: PDB / ID: 3jao
TitleCiliary microtubule doublet
Components
  • Tubulin alpha 1A chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / tubulin / microtubule doublet / cilia
Function / homology
Function and homology information


microtubule-based process / structural constituent of cytoskeleton / microtubule / GTPase activity / GTP binding
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
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GUANOSINE-5'-TRIPHOSPHATE / Tubulin alpha chain / Tubulin beta chain
Similarity search - Component
Biological speciesTetrahymena thermophila (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 23 Å
AuthorsMaheshwari, A. / Obbineni, J.M. / Bui, K.H. / Shibata, K. / Toyoshima, Y.Y. / Ishikawa, T.
CitationJournal: Structure / Year: 2015
Title: α- and β-Tubulin Lattice of the Axonemal Microtubule Doublet and Binding Proteins Revealed by Single Particle Cryo-Electron Microscopy and Tomography.
Authors: Aditi Maheshwari / Jagan Mohan Obbineni / Khanh Huy Bui / Keitaro Shibata / Yoko Y Toyoshima / Takashi Ishikawa /
Abstract: Microtubule doublet (MTD) is the main skeleton of cilia/flagella. Many proteins, such as dyneins and radial spokes, bind to MTD, and generate or regulate force. While the structure of the ...Microtubule doublet (MTD) is the main skeleton of cilia/flagella. Many proteins, such as dyneins and radial spokes, bind to MTD, and generate or regulate force. While the structure of the reconstituted microtubule has been solved at atomic resolution, nature of the axonemal MTD is still unclear. There are a few hypotheses of the lattice arrangement of its α- and β-tubulins, but it has not been described how dyneins and radial spokes bind to MTD. In this study, we analyzed the three-dimensional structure of Tetrahymena MTD at ∼19 Å resolution by single particle cryo-electron microscopy. To identify α- and β-tubulins, we combined image analysis of MTD with specific kinesin decoration. This work reveals that α- and β-tubulins form a B-lattice arrangement in the entire MTD with a seam at the outer junction. We revealed the unique way in which inner arm dyneins, radial spokes, and proteins inside MTD bind and bridge protofilaments.
History
DepositionJun 18, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 5, 2015Provider: repository / Type: Initial release
Revision 1.1Sep 16, 2015Group: Database references
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_image_scans / em_imaging_optics / em_software
Item: _em_imaging_optics.energyfilter_name / _em_software.image_processing_id
Revision 1.3Feb 21, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_conn_angle / pdbx_struct_oper_list / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.value / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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  • Biological unit as representative helical assembly
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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
  • EMDB-6312
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Tubulin alpha 1A chain
B: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,1086
Polymers100,0152
Non-polymers1,0934
Water1448
1
A: Tubulin alpha 1A chain
B: Tubulin beta chain
hetero molecules
x 115


Theoretical massNumber of molelcules
Total (without water)11,627,421690
Polymers11,501,726230
Non-polymers125,695460
Water4,143230
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
helical symmetry operation114
2


  • Idetical with deposited unit
  • helical asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Tubulin alpha 1A chain


Mass: 50107.238 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999 / Source method: isolated from a natural source / Source: (natural) Tetrahymena thermophila (eukaryote) / Strain: SB210 / References: UniProt: A0A0M3KKT1*PLUS
#2: Protein Tubulin beta chain


Mass: 49907.770 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999 / Source method: isolated from a natural source / Source: (natural) Tetrahymena thermophila (eukaryote) / Strain: SB210 / References: UniProt: A0A0M3KKT2*PLUS

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Non-polymers , 4 types, 12 molecules

#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.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-G2P / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


Mass: 521.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H18N5O13P3 / Comment: GMP-CPP, energy-carrying molecule analogue*YM
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsMICROTUBULES FROM TETRAHYMENA THERMOPHILA WERE IMAGED, BUT PROTEIN SEQUENCES FROM SUS SCROFA (UNP ...MICROTUBULES FROM TETRAHYMENA THERMOPHILA WERE IMAGED, BUT PROTEIN SEQUENCES FROM SUS SCROFA (UNP P02550 AND P02554) WERE USED FOR MODELING.

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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: microtubule doublet extracted from Tetrahymena cilia / Type: COMPLEX
Buffer solutionName: HMDEK buffer / pH: 7.4
Details: 30 mM HEPES, pH 7.4, 5 mM MgSO4, 1 mM DTT, 0.5 mM EDTA, 25 mM KCl, 4 mM CaCl2
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 300 mesh copper grid with holey carbon film, glow discharged
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Temp: 90 K / Humidity: 90 %
Details: Blot for 3 seconds in humid air atmosphere before plunging into liquid ethane (FEI VITROBOT MARK II).
Method: Blot for 3 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Oct 25, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 67000 X / Calibrated magnification: 67000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1000 nm / Cs: 1.5 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Temperature: 90 K / Temperature (max): 100 K / Temperature (min): 80 K
Image recordingElectron dose: 30 e/Å2 / Film or detector model: GENERIC CCD / Details: Gatan Ultrascan 4000
EM imaging opticsEnergyfilter name: Gatan GIF Tridem Energy Imaging Filter / Energyfilter upper: 25 eV / Energyfilter lower: 0 eV
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2SPIDER3D reconstruction
CTF correctionDetails: each particle
3D reconstructionMethod: R-weighted back projection / Resolution: 23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10700 / Nominal pixel size: 4.4776 Å / Actual pixel size: 4.4776 Å / Details: gold standard / Refinement type: HALF-MAPS REFINED INDEPENDENTLY / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: METHOD--kinesin binding, gradient density map analysis, and cross-correlation coefficient. The fitting for protofilament A10 as represented by BIOMT records 51-55 is ambiguous as it has been ...Details: METHOD--kinesin binding, gradient density map analysis, and cross-correlation coefficient. The fitting for protofilament A10 as represented by BIOMT records 51-55 is ambiguous as it has been assigned using only the cross-correlation coefficient. REFINEMENT PROTOCOL--RIGID BODY DETAILS--rigid body fitting of individual dimers
Atomic model buildingPDB-ID: 3J6E

3j6e


Accession code: 3J6E / Source name: PDB / Type: experimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms6700 0 66 8 6774

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