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- EMDB-8321: Near-atomic resolution cryo-EM reconstruction of doubly bound Tax... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-8321 | ||||||||||||
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Title | Near-atomic resolution cryo-EM reconstruction of doubly bound Taxol- and peloruside-stabilized microtubule | ||||||||||||
![]() | Peloruside- and taxol-bound microtubules | ||||||||||||
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![]() | Taxol / peloruside / microtubule / STRUCTURAL PROTEIN | ||||||||||||
Function / homology | ![]() 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 / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Separation of Sister Chromatids / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / COPI-mediated anterograde transport / 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 | ||||||||||||
Biological species | ![]() ![]() | ||||||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.5 Å | ||||||||||||
![]() | Kellogg EH / Nogales E | ||||||||||||
Funding support | ![]() ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 482.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 19.3 KB 19.3 KB | Display Display | ![]() |
Images | ![]() | 452.4 KB | ||
Filedesc metadata | ![]() | 6.9 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 5syeMC ![]() 8320C ![]() 8322C ![]() 8323C ![]() 5sycC ![]() 5syfC ![]() 5sygC C: citing same article ( M: atomic model generated by this map |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Peloruside- and taxol-bound microtubules | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.32 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Ternary complex of Taxol- and peloruside-stabilized microtubule l...
Entire | Name: Ternary complex of Taxol- and peloruside-stabilized microtubule lattice, alpha-tubulin, and beta-tubulin |
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Components |
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-Supramolecule #1: Ternary complex of Taxol- and peloruside-stabilized microtubule l...
Supramolecule | Name: Ternary complex of Taxol- and peloruside-stabilized microtubule lattice, alpha-tubulin, and beta-tubulin type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: Tubulin alpha chain
Macromolecule | Name: Tubulin alpha chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 48.679051 KDa |
Sequence | String: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLISQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRTIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GV UniProtKB: Tubulin alpha chain |
-Macromolecule #2: Tubulin beta chain
Macromolecule | Name: Tubulin beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 47.825859 KDa |
Sequence | String: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY Q UniProtKB: Tubulin beta chain |
-Macromolecule #3: GUANOSINE-5'-TRIPHOSPHATE
Macromolecule | Name: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: GTP |
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Molecular weight | Theoretical: 523.18 Da |
Chemical component information | ![]() ChemComp-GTP: |
-Macromolecule #4: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #5: GUANOSINE-5'-DIPHOSPHATE
Macromolecule | Name: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: GDP |
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Molecular weight | Theoretical: 443.201 Da |
Chemical component information | ![]() ChemComp-GDP: |
-Macromolecule #6: TAXOL
Macromolecule | Name: TAXOL / type: ligand / ID: 6 / Number of copies: 1 / Formula: TA1 |
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Molecular weight | Theoretical: 853.906 Da |
Chemical component information | ![]() ChemComp-TA1: |
-Macromolecule #7: Peloruside A
Macromolecule | Name: Peloruside A / type: ligand / ID: 7 / Number of copies: 1 / Formula: POU |
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Molecular weight | Theoretical: 548.663 Da |
Chemical component information | ![]() ChemComp-POU: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | helical reconstruction |
Aggregation state | helical array |
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Sample preparation
Concentration | 0.4 mg/mL | ||||||||||||||||||
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Buffer | pH: 6.8 Component:
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Grid | Model: Protochips CF-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV Details: Blot for 4 seconds, blot force 10, before plunging into liquid ethane (FEI VITROBOT MARK IV).. |
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Electron microscopy
Microscope | FEI TITAN |
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Temperature | Min: 93.0 K / Max: 93.0 K |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-20 / Number grids imaged: 1 / Number real images: 312 / Average exposure time: 0.3 sec. / Average electron dose: 8.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 50.0 µm / Calibrated defocus max: 3.8000000000000003 µm / Calibrated defocus min: 1.1 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 27500 |
Sample stage | Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER Cooling holder cryogen: NITROGEN |
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Image processing
Final reconstruction | Applied symmetry - Helical parameters - Δz: 9.43 Å Applied symmetry - Helical parameters - Δ&Phi: -27.7 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.09) / Number images used: 17069 |
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Segment selection | Number selected: 27275 Details: Microtubule filaments were manually selected using manualpicker.py. |
Startup model | Type of model: OTHER Details: Low-pass filtered reference model of microtubules decorated with kinesin |
Final angle assignment | Type: NOT APPLICABLE / Software - Name: FREALIGN (ver. 9.09) |
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Target criteria: correlation coefficient |
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Output model | ![]() PDB-5sye: |