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- PDB-3j6g: Minimized average structure of microtubules stabilized by taxol -

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Basic information

Entry
Database: PDB / ID: 3j6g
TitleMinimized average structure of microtubules stabilized by taxol
Components
  • Tubulin alpha-1A chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / microtubule / taxol
Function / homology
Function and homology information


Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / mitotic cell cycle / 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 / TAXOL / Tubulin alpha-1A chain / Tubulin beta chain
Similarity search - Component
Biological speciesSus scrofa (pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.5 Å
AuthorsAlushin, G.M. / Lander, G.C. / Kellogg, E.H. / Zhang, R. / Baker, D. / Nogales, E.
CitationJournal: Cell / Year: 2014
Title: High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis.
Authors: Gregory M Alushin / Gabriel C Lander / Elizabeth H Kellogg / Rui Zhang / David Baker / Eva Nogales /
Abstract: Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is ...Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is inhibited by anticancer agents like Taxol. We provide insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7-5.6 Å) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α-tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α- and β-tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces.
History
DepositionFeb 19, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 4, 2014Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Author supporting evidence / Data collection / Category: em_single_particle_entity / em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.2Feb 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 / struct_conn / struct_ref_seq_dif / 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 / _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_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-5897
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Tubulin alpha-1A chain
B: Tubulin beta chain
C: Tubulin alpha-1A chain
D: Tubulin beta chain
E: Tubulin alpha-1A chain
F: Tubulin beta chain
G: Tubulin alpha-1A chain
H: Tubulin beta chain
I: Tubulin alpha-1A chain
J: Tubulin beta chain
K: Tubulin alpha-1A chain
L: Tubulin beta chain
M: Tubulin alpha-1A chain
N: Tubulin beta chain
O: Tubulin alpha-1A chain
P: Tubulin beta chain
Q: Tubulin alpha-1A chain
R: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)887,00054
Polymers870,39818
Non-polymers16,60136
Water64936
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
DetailsThe 3 x 3 lattice represented in this entry is a segment of a microtubule.

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Components

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Protein , 2 types, 18 molecules ACEGIKMOQBDFHJLNPR

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


Mass: 48769.988 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02550
#2: Protein
Tubulin beta chain / Beta-tubulin


Mass: 47940.945 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02554

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Non-polymers , 5 types, 72 molecules

#3: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 9 / 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.: 9 / 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.: 9 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#6: Chemical
ChemComp-TA1 / TAXOL / Paclitaxel


Mass: 853.906 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 36 / Source method: isolated from a natural source / Formula: H2O

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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 stabilized by taxol / Type: COMPLEX
Buffer solutionName: 80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 1 mM DTT, 0.05% Nonidet P-40
pH: 6.8
Details: 80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 1 mM DTT, 0.05% Nonidet P-40
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 400 mesh C-flat 1.2/1.3, glow discharged in Edwards carbon evaporator
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Temp: 90.4 K / Humidity: 90 %
Details: The grid was blotted for 2 seconds before plunging into liquid ethane (FEI VITROBOT MARK II).
Method: The grid was blotted for 2 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: May 29, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 72000 X / Calibrated magnification: 72000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Specimen holder type: Gatan 626 holder / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 25 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 149
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1Rosettamodel fitting
2EMAN23D reconstruction
3FREALIGN3D reconstruction
4SPARX3D reconstruction
CTF correctionDetails: ctftilt
3D reconstructionMethod: projection matching / Resolution: 5.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24357 / Nominal pixel size: 1.74 Å / Actual pixel size: 1.74 Å / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: REFINEMENT PROTOCOL--flexible fitting DETAILS--Structure represents the minimized average structure of the 1% lowest energy structures from the refinement run.
Atomic model buildingPDB-ID: 1JFF
Accession code: 1JFF / Source name: PDB / Type: experimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms60318 0 1107 36 61461

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