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- PDB-5syf: High-resolution cryo-EM reconstruction of Taxol-stabilized microtubule -

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Basic information

Entry
Database: PDB / ID: 5syf
TitleHigh-resolution cryo-EM reconstruction of Taxol-stabilized microtubule
Components
  • Tubulin alpha chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / Taxol / microtubule
Function / homology
Function and homology information


Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling ...Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / Recruitment of NuMA to mitotic centrosomes / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / COPI-independent Golgi-to-ER retrograde traffic / MHC class II antigen presentation / COPI-mediated anterograde transport / microtubule-based process / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / microtubule / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal ...Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / 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 / Helix Hairpins / Rossmann fold / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / Tubulin alpha chain / Tubulin beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesSus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKellogg, E.H. / Nogales, E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM51487 United States
CitationJournal: J Mol Biol / Year: 2017
Title: Insights into the Distinct Mechanisms of Action of Taxane and Non-Taxane Microtubule Stabilizers from Cryo-EM Structures.
Authors: Elizabeth H Kellogg / Nisreen M A Hejab / Stuart Howes / Peter Northcote / John H Miller / J Fernando Díaz / Kenneth H Downing / Eva Nogales /
Abstract: A number of microtubule (MT)-stabilizing agents (MSAs) have demonstrated or predicted potential as anticancer agents, but a detailed structural basis for their mechanism of action is still lacking. ...A number of microtubule (MT)-stabilizing agents (MSAs) have demonstrated or predicted potential as anticancer agents, but a detailed structural basis for their mechanism of action is still lacking. We have obtained high-resolution (3.9-4.2Å) cryo-electron microscopy (cryo-EM) reconstructions of MTs stabilized by the taxane-site binders Taxol and zampanolide, and by peloruside, which targets a distinct, non-taxoid pocket on β-tubulin. We find that each molecule has unique distinct structural effects on the MT lattice structure. Peloruside acts primarily at lateral contacts and has an effect on the "seam" of heterologous interactions, enforcing a conformation more similar to that of homologous (i.e., non-seam) contacts by which it regularizes the MT lattice. In contrast, binding of either Taxol or zampanolide induces MT heterogeneity. In doubly bound MTs, peloruside overrides the heterogeneity induced by Taxol binding. Our structural analysis illustrates distinct mechanisms of these drugs for stabilizing the MT lattice and is of relevance to the possible use of combinations of MSAs to regulate MT activity and improve therapeutic potential.
History
DepositionAug 11, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 1, 2017Provider: repository / Type: Initial release
Revision 1.1Mar 8, 2017Group: Database references
Revision 1.2Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.3Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 6, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

Movie
  • Biological unit as representative helical assembly
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-8322
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  • Superimposition on EM map
  • EMDB-8322
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)98,3506
Polymers96,5052
Non-polymers1,8454
Water0
1
A: Tubulin alpha chain
B: Tubulin beta chain
hetero molecules
x 26


Theoretical massNumber of molelcules
Total (without water)2,557,087156
Polymers2,509,12852
Non-polymers47,959104
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
helical symmetry operation25
2


  • Idetical with deposited unit in distinct coordinate
  • helical asymmetric unit
TypeNameSymmetry operationNumber
helical symmetry operation1
3


  • Idetical with deposited unit
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 26 / Rise per n subunits: 9.48 Å / Rotation per n subunits: -27.7 °)

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Tubulin alpha chain


Mass: 48679.051 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: B6A7R0, UniProt: Q2XVP4*PLUS
#2: Protein Tubulin beta chain / Beta-tubulin


Mass: 47825.859 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02554

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Non-polymers , 4 types, 4 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.: 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
#6: Chemical ChemComp-TA1 / TAXOL / Paclitaxel


Mass: 853.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Ternary complex of Taxol-stabilized microtubule lattice, alpha-tubulin, and beta-tubulin
Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Sus scrofa (pig)
Buffer solutionpH: 6.8
Buffer component
IDConc.NameFormulaBuffer-ID
180 mMPIPESC8H18N2O6S21
21 mMmagnesium chlorideMgCl21
31 mMEGTAC14H24N2O101
41 mMDTTC4H10O2S21
50.05 % volNP-40octylphenoxypolyethoxyethanol1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Protochips CF-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: Blot for 4 seconds, blot force 10, before plunging into liquid ethane (FEI VITROBOT MARK IV).

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 27500 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1100 nm / Calibrated defocus max: 3800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Temperature (max): 93 K / Temperature (min): 93 K
Image recordingAverage exposure time: 0.3 sec. / Electron dose: 8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 312
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 20 / Used frames/image: 1-20

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Processing

EM software
IDNameVersionCategoryDetails
2Leginon3.2image acquisitionLeginon was used to collect images.
3Appion3.2image acquisitionAppion was used to store and do preliminary image processing.
5CTFFIND4CTF correction
8UCSF Chimera1.1model fitting
10Rosetta3.6model refinement
11REFMAC5.5model refinement
12EMAN22.07initial Euler assignment
13FREALIGN9.09final Euler assignment
15FREALIGN9.093D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -27.7 ° / Axial rise/subunit: 9.48 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 27275
Details: Microtubule filaments were manually selected using manualpicker.py.
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17069 / Algorithm: FOURIER SPACE / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: correlation coefficient

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